Get  the  Best 


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DORLAND'S 


MEL 


A  New  and 
Dentistry, 
new  and 
of  Bacilli 
Operation 
ment,  etc 
Pocket  M 
full  flexifc 

JUST  issue: 
It  conti 


This  book 
tionary,  and  y 
finest  quality,  i 
and  is  just  the 
stant  reference 
defines  hundret 
is  especially  fu 
great  practical 
Anew  feature 
them  in  colors, 

"  I  must  ackn 
tively  small  space 
finding  some  of  tl 
Professor  of  Prin 


"  Dr.  Dorland 
size.     No  errors 
cology,  Johns  Hopkins  Umvei  sity,  Baltimore. 


MEDICAL    >SC1HI©(DL 
LHEHBAIEY 


Gift  of 
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iary.  It 
ldred  of 
lethods. 
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Fourth  Edition,  Enlarged  With  Complete  Vocabulary 

THE 

AMERICAN  POCKET 

MEDICAL  DICTIONARY 

EDITED  BY 

W.  A.  NEWMAN   DORLAND,  A.  M.,  M.  D., 

Assistant    Demonstrator  of   Obstetrics,   University   of    Pennsylvania. 

FOURTH  EDITION,  REVISED    RECENTLY  ISSUED. 

Bound  in  Full  Leather,  Limp,  with  Gold  Edges.    Price,  $1.00  neti 
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sion of  any  old  work,  but  it  has  been  written  entirely  anew 
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of  modern  medicine.  This  makes  an  unusually  large  vocabu- 
lary. Besides  the  ordinary  dictionary  terms  the  book  contains 
a  wealth  of  anatomical  and  other  tables.  This  matter  is 
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terior. I  can  recommend  it  to  our  students  without  reserve." — J  AMES  W.  Hol- 
land, M.  D.,  of  Jefferson  Medical  College. 

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ESSENTIALS 


OF 


BACTERIOLOGY 


Since  the  issue  of  the  first  volume  of  the 
Saunders  Question=Compends, 

OVER  250,000  COPIES 

of  these  unrivalled  publications  have  been  sold. 
This  enormous  sale  is  indisputable  evidence 
of  the  value  of  these  self-helps  to  students 
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Ocular 


Nose-piece 


Coarse  Adjustment 
Fine  Adjustment 


BACTERIOLOGICAL  MICROSCOPE  (with  Abbe  and  Blender  in  Position). 


SAUNDERS'  QUESTION-COMPENDS,  No.  20 


ESSENTIALS 


BACTERIOLOGY 


CONCISE  AND  SYSTEMATIC  INTRODUCTION 
TO  THE  STUDY  OF  MICRO-ORGANISMS 

/? 
vCbv 

M.  V.   BALL,  M.D. 

Formerly  Resident  Physician,  German  Hospital,  Philadelphia ; 
formerly  Bacteriologist  to  St.  Agnes'  Hospital 

FIFTH  EDITION,  THOROUGHLY  REVISED 

BY 

KARL  M,  VOGEL,  M.D. 

Assistant  in  Pathology,  College  of  Physicians  and  Surgeons, 
Columbia  University,  New  York  City 

With    Ninety-Six    Illustrations 
some  in  Colors,  and  Six  Plates 


PHILADELPHIA  AND   LONDON 

W.  B.  SAUNDERS   &   COMPANY 

1905 


Set  up,  electrotyped,  printed,  and  copyrighted  October,  1891.     Reprinted  October, 

1892.     Revised,  reprinted,  and  recopyrighted  May,  1893.     Reprinted  June, 

1894.    Revised,  reprinted,  and  recopyrighted  November,  1896.    Reprinted 

October,    1898.     Revised,   reprinted,    and   recopyrighted    March, 

1900.     Reprinted    May,    1903.     Revised,   reprinted, 

and   recopyrighted    August,    1904. 


Copyright,  1904,  by  W.  B.  Saunders  &  Company. 


Reprinted  October,  1905. 


ELECTROTYPED    BY 
WESTCOTT  &  THOMSON,   PHILAOA. 


PRESS    OF 
W.   B.   SAUNDERS  &  COMPANY. 


1165- 


PREFACE  TO  THE  FIFTH  EDITION 


The  progress  in  bacteriology  during  the  last  few  years 
has  involved  more  or  less  radical  changes  in  many  depart- 
ments of  the  science.  Recent  work  on  such  subjects  as 
immunity,  tuberculosis,  dysentery,  yellow  fever,  the  bu- 
bonic plague,  and  other  infectious  diseases  has  rendered 
obsolete  many  portions  of  any  but  the  most  modern  books, 
and  in  countless  minor  details  the  teaching  of  to-day  differs 
from  that  of  even  a  few  years  ago. 

In  this  revision  the  attempt  has  been  made  to  reflect  as 
faithfully  as  possible  the  present  status  of  bacteriology 
without  overstepping  the  limits  set  by  the  scope  of  a  book 
intended  primarily  as  *an  aid  to  students.  Much  assist- 
ance has  been  derived  from  the  "  Manual  of  Bacteriol- 
ogy" of  Muir  and  Ritchie,  and  from  F.  C.  Wood's 
"Laboratory  Guide  to  Clinical  Pathology." 

7 


5506r() 


PKEFAOE  TO  FIRST  EDITION. 


Feeling  the  need  of  a  Compendium  on  the  subject  of 
this  work,  it  has  been  our  aim  to  produce  a  concise  treatise 
upon  the  Practical  Bacteriology  of  to-day,  chiefly  for  the 
medical  student,  which  he  may  use  in  his  laboratory. 

It  is  the  result  of  experience  gained  in  the  Laboratory 
of  the  Hygienical  Institute,  Berlin,  under  the  guidance  of 
Koch  and  Frankel ;  and  of  information  gathered  from  the 
original  works  of  other  German,  as  well  as  of  French, 
bacteriologists. 

Theory  and  obsolete  methods  have  been  slightly  touched 
upon.  The  scope  of  the  work,  and  want  of  space,  forbade 
adequate  consideration  of  them.  The  exact  measurements 
of  bacteria  have  not  been  given.  The  same  bacterium 
varies  often  much  in  size,  owing  to  differences  in  the  media, 
staining,  etc. 

We  have  received  special  help  from  the  following  books, 
which  we  recommend  to  students  for  further  reference : — 

Mac6:    Trait6  pratique  de  Bacteriologie. 
Frankel:    Grundriss  der  Bakterienkunde. 
Eisenberg:    Bakteriologische  Diagnostik. 
Crookschank,  E.  M.:    Manual  of  Bacteriology. 
Gunther:    Einflihring  in  das  Studium  der  Bacteriologie,  etc. 
Woodhead  and  Hare:    Pathological  Mycology. 
Salmonsen:    Bacteriological  Technique  (English  translation). 

M.  V.  BALL. 

Buffalo,  N.  Y.,  October  1, 1891. 
62  Delaware  Avenue. 


CONTENTS 


PART  I. 
GENERAL  CONSIDERATIONS  AND  TECHNIQUE. 


PAGE 


Introduction 15 

Chapter  I.— Classification,  Structure,  and  Reproduction.  17 

II.— Origin,  Life,  Growth,  and  Properties  ....  23 

"     III.— Methods  of  Examination 26 

"      IV.— Staining  of  Bacteria 30 

V— General  Method  of  Staining  Specimens  ...  35 

"      VI.— Special  Methods  of  Staining 39 

"    VII.— Methods  of  Culture 42 

"  VIIL— Nutrient  Media 48 

M     IX.- -Solid  Transparent  Media     52 

"       X.— Inoculation  of  Gelatine  and  Agar 60 

"     XL— Growth  and  Appearances  of  Colonies    ...  64 

"    XII. — Cultivation  of  Anaerobic  Bacteria 67 

"  XIII.— Infection 70 

"  XIV.— Immunity 72 

"    XV.— Animal  Experiments 76 


PART  II. 

SPECIAL  BACTERIOLOGY. 

Chapter  I.— Non-Pathogenic  Bacteria 80 

Bacillus  Prodigiosus 80 

Indicus 81 

9 


10  CONTENTS. 

PAGE 

Bacillus — 

Mesentericus  Vulgatus 81 

Megateriuni 82 

Ramosus     82 

Bacterium  Zopfi 83 

Bacillus  Subtilia 83 

Spinosus 84. 

Some  Bacteria  in  Milk 84 

Bacillus  Acidi  Lactici      .    .    .  84 

Butyricus 85 

Amylobacter      85 

Lactis  Cyanogenus 86 

Lactis  Erythrogenes 86 

Examination  of  Milk  in  Stained  Specimens 87 

Some  Non-Pathogenic  Bacteria  of  Water     87 

Bacillus  Violaceus * 87 

Cceruleus 88 

*         Fluorescent  Bacteria 88 

Phosphorescent  Bacteria 89 

Leptothrix,    Crenothrix,    Cladothrix,    and 

Beggiatoa 90 

Micro-organisms  found  in  Urine 91 

Spirillum 92 

Rubrum;  Concentricum 92 

Sarcina 92 

Lutea 92 

Aurantica  Flava,  Rosea,  and  Alba 93 

Ventriculi , 93 

s-Oppler  Bacillus 93 


Chapter  II. — Pathogenic  Bacteria •  .  94 

Bacteria  Pathogenic  for  Man  and  Other  Animals  ...  94 

Bacillus  Anthracis 94 

•  Tuberculosis 97 

Lepra  Bacillus 107 

Syphilis  Bacillus 108 

Bacillus  of  Glanders 108 


CONTENTS.  11 

PAGE 

Bacillus — 

of  Diphtheria HO 

pseudo- 114 

of  Typhoid  Fever 115 

Paracolon  or  Paratyphoid .    .    .   121 

Psittacosis       121 

Coli  Communis 122 


Chapter  III. — Pathogenic  Bacteria — continued  .   .   .   .   .   124 

Spirillum  Cholera 124 

Bacteria  Similar  to  Spirillum  Cholerae   ........   127 

Finkler-Prior     .   T\    .'.    .    ...    r  ..*...    .    .   127 

Tyrogenum 128 

Vibrio  Metschnikovi 129 

Bacteria  of  Pneumonia 130 

Pneumobacillus  of  Friedlander ' .    .   131 

ofFrankel 132 

Antitoxin  of  Pneumonia 134 

Bacillus  of  Rhinoscleroma 135 

Diplococcus  Intracellularis  Meningitidis 135 

Micrococcus  Tetragenus 135 

Capsule  Bacillus 136 

Bacillus  of  Influenza 137 

Micro-organisms  of  Suppuration ,   137 

Streptococcus  Pyogenes 138 

Staphylococcus  Pyogenes  Aureus 140 

,    Pyogenes  Albus 141 

Micrococcus  Pyogenes  Citreus     .........   141 

Cereus  Albus 141 

Cereus  Flavus 141 

Pyogenes  Tenuis 142 

Bacillus  Pyocyaneus 142 

Micrococcus  Gonorrhoeae 143 

Microbes  Similar  to  Gonorrhoea 145 

Bacillus  of  Tetanus 147 


12  CONTENTS. 

PAGE 

Bacillus — 

(Edematis  Maligni    .............  150 

Spirillum  of  Relapsing  Fever 152 

Bacillus  of  Soft  Chancre 153 

Icteroides    ........  154 

of  Bubonic  Plague 155 

#                      of  Dysentery      157 

Aerogenes  Capsulatus 158 

Micrococcus  Melitensis 159 

Pathogenic  Protozoa . 159 

Malarial  Parasite 159 

Amoeba  Dysenterise 163 

Small-pox  and  Vaccinia  . 163 

Trypanosomes 163 

Chapter  IV. — Bacteria    Pathogenic  for  Animals,   but 

not  for  Man 164 

Bacillus  of  Symptomatic  Anthrax 164 

of  Chicken  Cholera  .    .    . 165 

Bacteria  of  Hemorrhagic  Septicaemia,  Swine  Plague, 

Duck  Cholera,  etc 166 

Bacillus  of  Erysipelas  of  Swine 167 

Murisepticus 168 

Micrococcus  of  Mai  de  Pis 169 

Bacillus  Alvei 169 

Micrococcus  Amylovorus 170 

Bacterium  Termo 170 

Proteus  Vulgaris 170 

Mirabilis 171 

Zenkeri 0   .......   .  171 


APPENDIX. 


Yeasts 173 

Oidiums 174 

Moulds 175 


CONTENTS.  13 

PAGE 

Cladothrices  and  Strep tothrices 177 

Streptothrix,  or  Cladothrix  Actinomyces  (Ray  Fungus)  .    .  177 

Madura    .    . 178 

Farcinica 179 

Examination  of  Air 180 

of  Water 184 

of  Soil 187 

Bacteria  of  Milk  and  Other  Foods 188 

Examination  of  the  Organs  and  Cavities  of  the  Human 

Body 189 

Tables  of  Chief  Characteristics  of  the  Principal  Bacteria  .    .  194 

Part    I.  Non-Pathogenic    . 194 

Part  II.  Pathogenic 218 


INTRODUCTION 


History. — The  microscope  was  invented  about  the  latter 
part  of  the  sixteenth  century ;  and  soon  after,  by  its  aid, 
minute  organisms  were  found  in  decomposing  substances. 
Kircher,  in  1646,  suggested  that  diseases  might  be  due  to 
similar  organisms ;  but  the  means  at  his  disposal  were  in- 
sufficient to  enable  him  to  prove  his  theories.  Anthony 
Van  Leuwenhoeck,  of  Delft,  Holland  (1680  to  1723),  so 
improved  the  instrument  that  he  was  enabled  thereby  to 
discover  micro-organisms  in  vegetable  infusion,  saliva,  fecal 
matter,  and  scrapings  from  the  teeth.  He  distinguished 
several  varieties,  showed  them  to  have  the  power  of  loco- 
motion, and  compared  them  in  size  with  various  grains  of 
definite  measurement.  It  was  a  great  service  that  this 
"Dutch  naturalist"  rendered  the  world;  and  he  can  rightly 
be  called  the  "  father  of  microscopy." 

Various  theories  were  then  formulated  by  physicians  to 
connect  the  origin  of  different  diseases  with  bacteria;  but  no 
proofs  of  the  connection  could  be  obtained.  Andry,  in  1701, 
called  bacteria  worms.  Miiller,  of  Copenhagen,  in  1786,  made 
a  classification  composed  of  two  main  divisions — monas  and 
vibrio  ;  and  with  the  aid  of  the  compound  microscope  was 
better  able  to  describe  them.  Ehrenberg,  in  1833,  with  still 
better  instruments,  divided  bacteria  into  four  orders:  bac- 
terium, vibrio,  spirillum,  and  spirochete.  It  was  not  until 
1863  that  any  positive  advance  was  made  in  connecting 
bacteria  with  disease.      Rayer  and  Davaine   had   in   1850 

15- 


16 


INTRODUCTION 


already  found  a  rod-shaped  bacterium  in  the  blood  of  ani. 
mals  suffering  from  splenic  fever  {sang  de  rate),  but  they 
attached  no  special  significance  to  their  discovery  until 
Pasteur  made  public  his  grand  researches  in  regard  to  fer- 
mentation and  the  role  bacteria  played  in  the  economy. 
Then  Davaine  resumed  his  studies,  and  in  1863  established 
by  experiments  the  bacterial  nature  of  splenic  fever  or  an- 
thrax. 

But  the  first  complete  study  of  a  contagious  affection  was 
made  by  Pasteur  in  1869,  in  the  diseases  affecting  silk-worms 
— pebrine  and  flacherie — which  he  showed  to  be  due  to  micro- 
organisms. 

Then  Koch,  in  1875,  described  more  fully  the  anthrax 
bacillus,  gave  a  description  of  its  spores  and  the  properties 
of  the  same,  and  was  enabled  to  cultivate  the  germ  on  arti- 
ficial media ;  and,  to  complete  the  chain  of  evidence,  Pas- 
teur and  his  pupils  supplied  the  last  link  by  reproducing  the 
same  disease  in  animals  by  artificial  inoculation  from  pure 
cultures.  The  study  of  the  bacterial  nature  of  anthrax  has 
been  the  basis  of  our  knowledge  of  all  contagious  maladies, 
and  most  advances  have  been  made  first  with  the  bacterium 
of  that  disease. 

Since  then  bacteriology  has  grown  to  huge  proportions — 
become  a  science  in  itself — and  thousands  of  earnest  workers 
are  adding  yearly  solid  blocks  of  fact  to  the  structure,  which 
structure  it  will  be  our  aim  to  briefly  describe  in  the  pages 
which  are  to  follow. 


ESSENTIALS  OF  BACTERIOLOGY. 


PART  I. 
GENERAL  CONSIDERATIONS. 


CHAPTER  I. 

BACTERIA. 

The  bacteria  occupy  the  lowest  plane  of  plant  life  known  to 
us,  though  they  are  by  no  means  as  primitive  in  their  biology 
as  was  formerly  supposed,  and  it  is  quite  possible  that  still 
simpler  forms  may  be  discovered. 

The  numerous  unicellular  vegetable  organisms  which  form 
the  lower  limit  of  plant  life  as  we  know  it  multiply  by  fission 
and  are  hence  called  the  Schizophyta,  or  splitting  plants.  This 
group  is  subdivided  into  two  classes — (a)  the  Schizophycese,  or 
fission  alga?,  and  (b)  the  Schizomycetes,  or  fission  fungi,  or  bac- 
teria, as  we  usually  call  them. 

Lately  it  has  Become  customary  to  subdivide  the  bacteria 
themselves  somewhat  arbitrarily  into  two  classes — the  lower 
bacteria  and  the  higher  bacteria. 

The  lower  bacteria  are  unicellular  masses  of  protoplasm  of 
microscopic  size,  multiplying  by  fission  and  existing  without 
chlorophyll.  Three  main  types  are  found  :  (1)  Globular  forms, 
called  cocci ;  (2)  straight  rod-shaped  forms,  called  bacilli ;  (3) 
curved  or  spiral  rods,  called  spirilla. 

The  higher  bacteria  show  a  tendency  toward  a  more  com- 
plicated mode  of  organization  in  two  ways  :  (1)  They  consist  of 
filaments  made  up  of  separate  individuals,  but  which  exhibit 
enough  independence  to  foreshadow  the  rudiments  of  a  physi- 
2  (17) 


18         ESSENTIALS  OF  BACTERIOLOGY. 

ological  division  of  labor.     (2)  Certain  elements  may  be  differ- 
entiated for  the  purpose  of  reproduction. 

The  Staphylococcus  pyogenes,  the  anthrax  bacillus,  and  the 
spirillum  of  relapsing  fever  are  typical  forms  of  the  lower  bac- 
teria, while  the  actinomyces,  or  ray-fungus,  is  the  most  impor- 
tant pathogenic  member  of  the  higher  bacteria. 


Fig.  1. 


-V 


Micrococcus.  Spirillum.  Bacillus. 

Structure.  Bacteria  are  cells  ;  they  appear  as  round  or  cylin- 
drical of  an  average  diameter  or  transverse  section  of  0.001  mm. 
(=1  micromillimeter),  written  1  /*.  The  cell,  as  other  plant- 
cells,  is  composed  of  a  membranous  cell- wall  and  cell-contents  ; 
"  cell-nuclei "  can  in  some  cases  be  seen  by  the  use  of  special 
stains. 

Cell- Wall.  The  cell-wall  is  composed  either  of  plant  cellu- 
lose, or  a  form  of  albumin,  since  it  is  less  permeable  than  cellu- 
lose membrane.  The  membrane  is  firm,  and  can  be  brought 
plainly  into  view  by  the  action  of  iodin  upon  the  cell-contents, 
which  contracts  them. 

Cell-Contents.  The  contents  of  the  cell  consist  mainly  of 
protoplasm,  usually  homogeneous,  but  in  some  varieties,  finely 
granular,  or  holding  pigment,  chlorophyll,  fat-droplets,  and  sul- 
phur in  its  structure. 

It  is  composed  chiefly  of  mycoprotein. 

Gelatinous  Membrane.  The  outer  layer  of  the  cell-membrane 
can  absorb  water  and  become  gelatinoid,  forming  either  a  little 
envelope  or  capsule  around  the  bacterium  or  preventing  the 
separation  of  the  newly-branched  germs,  forming  chains  and 
bunches,  as  strepto-  and  staphylo-cocci.  Long  filaments  are  also 
formed. 

Zooglcea.    When  this  gelatinous  membrane  is  very  thick,  irre- 


BACTERIA 


19 


gular  masses  of  bacteria  will  be  tunned,  the  whole  growth  being 
in  one  jelly-like  lump.  This  is  termed  a  zooglcea  {$<jwv,  animal, 
yloios,  glue). 

Locomotion.  Many  bacteria  possess  the  faculty  of  self-move- 
ment, carrying  themselves  in  all  manner  of  ways  across  the 
microscopic  field,  some  very  quickly,  others  leisurely. 

Vibratory  Movements.  Some  bacteria  vibrate  in  themselves, 
appearing  to  move,  but  they  do  not  change  their  place ;  these 
movements  are  denoted  as  molecular  or  "  Broivnian"  and  are 
due  to  purely  physical  causes. 


Fig.  2. 


Zoogloea. 


Flagella.  Little  threads  or  lashes  are  found  attached  to  many 
of  the  motile  bacteria,  either  at  the  poles  or  along  the  sides, 
sometimes  only  one,  and  on  some  several,  forming  a  tuft. 

These  flagella  are  in  constant  motion  and  can  probably  be 
considered  as  the  organs  of  locomotion  ;  they  have  not  yet  been 
discovered  upon  all  the  motile  bacteria,  owing  no  doubt  to  our 
imperfect  methods  of  observation.  They  can  be  stained  and 
have  been  photographed.  See  Fig.  3.  Flagella  serve  some- 
times to  increase  food-supply,  and  have  been  found  on  some 
species  which  are  non-motile. 


20 


ESSENTIALS    OF    BACTERIOLOGY 


Reproduction.     Bacteria  multiply  through  simple  division  or 
fission  as  it  is  called.     Spore  formation  is  simply  a  resting  stage 

Fig.  3. 


Flagella. 

and  not  a  means  of  multiplication.     To  accomplish  division 
the  cell  elongates,  and  at  one  portion,  usually  the  middle,  the 

Fig.  4. 
1  2 


Division  of  a  Micrococcus.    (After  Mace\) 


-'TT^'N 


Division  of  a  Bacillus.    (After  Mace\) 

cell- wall  indents  itself  gradually,  forming  a  septum  and  dividing 
the  cell  into  two  equal  parts,  just  as  occurs  in  the  higher  plant 
and  animal  cells.     See  Fig.  4. 


BACTERIA. 


21 


Successive  divisions  take  place,  the  new  members  either  exist- 
ing as  separate  cells  or  forming  part  of  a  community  or  group. 
It  has  been  computed  that  if  division  takes  place  every  hour, 
as  it  often  does,  one  individual  in  twenty-four  hours  will  have 
17,000,000  descendants. 

Spore  Formations.  Two  forms  of  sporulation ,  Endosporous  and 
A rthrosporous.  First,  a  small  granule  develops  in  the  protoplasm 
of  a  bacterium,  this  increases  in  size,  or  several  little  granules 
coalesce  to  form  an  elongated,  highly  refractive,  clearly  defined 
object,  rapidly  attaining  its  real  size,  and  this  is  the  spore.  The 
remainder  of  the  cell-contents  has  now  disappeared,  leaving 
the  spore  in  a  dark,  very  resistant,  membrane  or  capsule,  and 
beyond  this  the  weak  cell-wall.  The  cell-wall  dissolves  gradu- 
ally or  stretches  and  allows  the  spore  to  be  set  free. 

Each  bacterium  gives  rise  to  but  one  spore.  It  may  be  at 
either  end  or  in  the  middle  (Fig.  5).  Some  rods  take  on  a  pecu- 
liar shape  at  the  site  of  the  spore,  making  the  rod  look  like  a 
drum-stick  or  spindle,  Clostridium  (Fig.  6). 


Fig.  6. 


Sporulation.     After  De  Bary. 


a 

Clostridium. 


Spore  Contents.  What  the  real  contents  of  spores  are  is  not 
known.  In  the  mother  cell  at  the  site  of  the  spore  little  gran- 
ules have  been  found  which  stain  differently  from  the  rest  of 
the  cell,  and  these  are  supposed  to  be  the  beginnings,  the  spwo- 


22         ESSENTIALS  OF  BACTERIOLOGY. 

genie  bodies.  The  most  important  part  of  the  spore  is  its  cap- 
sule; to  this  it  owes  its  resisting  properties.  It  consists  of  two 
separate  layers,  a  thin  membrane  around  the  cell,  and  a  firm 
outer  gelatinous  envelope. 

Germination.  AVhen  brought  into  favorable  conditions,  the 
spore  begins  to  lose  its  shining  appearance,  the  outer  firm  mem- 
brane begins  to  swell,  and  it  now  assumes  the  shape  and  size 
of  the  cell  from  which  it  sprang,  the  capsule  having  burst,  so  as 
to  allow  the  young  bacillus  to  be  set  free. 

Requisites  for  Spore  Formation.  It  was  formerly  thought  that 
when  the  substratum  could  no  longer  maintain  it,  or  had  become 
infiltrated  with  detrimental  products,  the  bacterium-cell  pro- 
duced spores,  or  rather  turned  itself  into  a  spore  to  escape  anni- 
hilation ;  but  we  believe  now  that  only  when  conditions  are  the 
most  favorable  to  the  well-being  of  the  cell,  does  it  produce 
fruit,  just  as  with  every  other  type  of  plant  or  animal  life,  a  cer- 
tain amount  of  oxygen  and  heat  being  necessary  for  good  spore 
formation.    The  question  is  still  unsettled,  however. 

Asporogenic  Bacteria.  Bacteria  can  be  so  damaged  that  they 
will  remain  sterile,  not  produce  any  spores.  This  condition  can 
be  temporary  only,  or  permanent. 

Arthrosporous.  All  the  above  remarks  relate  to  Endospores, 
spores  that  arise  within  the  cells. 

In  the  other  group  called  Arthrospores,  individual  members 
of  a  colony  or  aggregation  leave  the  same,  and  become  the  origi- 
nators of  new  colonies,  thus  assuming  toe  character  of  spores. 

The  Micrococci  furnish  examples  of  this  form. 

Some  authorities  have  denied  the  existence  of  the  arthro- 
sporous formation. 

Resistance  of  Spores.  Because  of  the  very  tenacious  envelope, 
the  spore  is  not  easily  influenced  by  external  measures.  It  is 
said  to  be  the  most  resisting  object  of  the  organic  world. 

Chemical  and  physical  agents  that  easily  destroy  other  life 
have  very  little  effect  upon  it. 

Many  spores  require  a  temperature  of  140°  C.  dry  heat  for 
several  hours  to  destroy  them.  The  spores  of  a  variety  of  potato- 
bacillus  (bacillus  mesentericus)  can  withstand  the  application  of 
steam  at  100°  C.  for  four  hours. 


ORIGIN    OP   BACTERIA.  23 

CHAPTER  II. 

ORIGIN   OP   BACTERIA   AND   THEIR  DISTRIBUTION. 

As  Pasteur  has  shown,  all  bacteria  develop  from  pre-existing 
bacteria,  or  the  spores  of  the  same.    They  cannot  arise  de  novo. 

The  wide  and  almost  universal  diffusion  of  bacteria  is  due  to 
the  minuteness  of  the  cells  and  the  few  requirements  for  their 
existence.     In  a  drop  of  water  1700  million  cocci  can  find  room. 

Very  few  places  are  free  from  germs  ;  the  air  on  the  high  seas, 
and  on  the  mountain  tops,  is  said  to  be  free  from  bacteria, 
but  it  is  questionable. 

One  kind  of  bacterium  will  not  produce  another  kind. 

A  bacillus  does  not  arise  from  a  micrococcus  or  the  typhoid 
fever  bacillus  produce  the  bacillus  of  tetanus. 

This  subject  has  been  long  and  well  discussed,  and  it  would 
take  many  pages  to  state  the  "  pros"  and  "  cons,"  therefore,  this 
positive  statement  is  made,  it  being  the  position  now  held  by  the 
principal  authorities. 

Saprophytes  and  Parasites.  {Saprophytes,  gd-n-pSg,  putrid.  <j>vt6v, 
plant.  Parasites,  napa,  aside  of,  airoq,  food.)  Those  bacteria 
which  live  on  the  dead  remains  of  organic  life  are  known  as 
Saprophytic  Bacteria,  and  those  which  choose  the  living  bodies 
of  their  fellow-creatures  for  their  habitat  are  called  Parasitic 
Bacteria.  Some,  however,  develop  equally  well  as  Saprophytes 
and  Parasites.     They  are  called  Facultative  Parasites. 

Conditions  of  Life  and  Growth  of  Bacteria.  Influence  of  Tem- 
perature.— In  general,  a  temperature  ranging  from  10°  C.  to  40° 
C.  is  necessary  to  their  life  and  growth. 

Saprophytes  take  the  lower  temperatures  ;  Parasites,  the  tem- 
perature more  approaching  the  animal  heat  of  the  warm-blooded. 
Some  forms  require  a  nearly  constant  heat,  growing  within  very 
small  limits,  as  the  Bacillus  of  Tuberculosis. 

Some  forms  can  be  arrested  in  their  development  by  a  warmer 
or  colder  temperature,  and  then  restored  to  activity  by  a  return 
to  the  natural  heat. 


24         ESSENTIALS  OF  BACTERIOLOGY. 

A  few  varieties  exist  only  at  freezing  point  of  water;  and 
others  again  will  not  live  under  a  temperature  of  60°  C. 

For  the  majority  of  Bacteria  a  temperature  of  60°  C.  is  de- 
structive ;  and  several  times  freezing  and  thawing  very  fatal. 

Influence  of  Oxygen. — Two  varieties  of  bacteria  in  relation  to 
oxygen.  The  one  terobie,  growing  in  air;  the  other,  anserobic, 
living  without  air. 

Obligate  serobins,  those  which  exist  only  when  oxygen  is  present. 

Facultative  asrobins,  those  that  live  best  when  oxygen  is  present, 
but  can  live  without  it. 

Obligate  or  true  anserobins,  those  which  cannot  exist  where 
oxygen  is.  Facultative  anwrobins,  those  which  exist  better  where 
there  is  »^  oxygen,  but  can  live  in  ito  prcQcnce.'*"1^^^'-^ 

Some  derive  the  oxygen  which  they  require  out  of  their  nutri- 
ment, so  that  a  bacterium  may  be  aerobic  and  yet  not  require 
the  presence  of  free  oxygen. 

iErobins  may  consume  the  free  oxygen  of  a  region  and  thus 
allow  the  anserobins  to  develop.  By  improved  methods  of  cul- 
ture many  varieties  of  anserobins  have  been  discovered. 

Influence  of  Light.— Sunlight  is  very  destructive  to  bacteria. 
A  few  hours'  exposure  to  the  sun  has  been  fatal  to  anthrax 
bacilli,  and  the  cultures  of  bacillus  tuberculosis.  The  sun's 
rays,  however,  must  come  in  direct  contact  with  the  germs,  and 
are  usually  only  active  on  the  surface-cultures.  The  rays  at 
the  violet  end  of  the  spectrum  are  the  most  active.  The 
electric  arc  light  has  much  the  same  effect  as  sunlight  on  bac- 
teria. 

Effects  of  Electricity. — Electricity  arrests  growth. 

Effects  of  R'dntgen  Rays. — Haye  little  or  no  effect  on  artificial 
cultures,  but  in  the  living  tissues  a  pronounced  bactericidal 
effect  is  produced,  perhaps  through  the  stimulation  of  the 
body-cells. 

Vital  Actions  of  Microbes.  Bacteria  feeding  upon  organic  com- 
pounds produce  chemical  changes  in  them,  not  only  by  the  with- 
drawal of  certain  elements,  but  also  by  the  excretion  of  these 
elements  changed  by  digestion.  Sometimes  such  changes  are 
destructive  to  themselves,  as  when  lactic  and  butyric  acids  are 
formed  in  the  media. 


ORIGIN    OF    BACTERIA.  25 

Oxidation  and  reduction  are  carried  on  by  some  bacteria.  Am- 
monia, hydrogen  sulphide,  and  trimethylamin  are  a  few  of  the 
chemical  products  produced  by  bacteria.  Nitrites  in  the  soil 
are  reduced  to  ammonia. 

Nitrification. — Albuminoids  changed  into  indol,  skatol,  leucin, 
etc. ;  then  these  into  ammonia.  Ammonia  into  nitrites.  Ni- 
trites into  nitrates. 

Ptomaines.  Brieger  found  a  number  of  complex  alkaloids, 
closely  resembling  those  found  in  ordinary  plants,  and  which 
he  named  ptomaines,  from  nrcbfia  (corpse),  because  obtained 
from  putrefying  objects. 

Proteins.  The  components  of  the  bacterial  cell  may  cause 
inflammation  and  fever. 

Putrefaction.  When  fermentation  is  accompanied  by  devel- 
opment of  offensive  gases  a  decomposition  occurs,  which  is 
called  putrefaction,  and  this,  in  organic  substances,  is  due 
entirely  to  bacteria. 

Producers  of  Disease.  Various  pathological  processes  are 
caused  by  bacteria,  the  name  given  to  such  diseases  being  in- 
fectious diseases,  and  the  germs  themselves  called  disease-pro- 
ducing or  pathogenic  bacteria.  Those  which  do  not  form  any 
pathological  process  are  called  non-pathogenic  bacteria. 

Ferments  are  diastatic,  changing  starch  into  sugar ;  proteolytic, 
transforming  albumins  into  more  soluble  substances;  gelatin 
liquefaction  is  an  example. 

Inverting,  changing  a  sugar  from  one  that  does  not  undergo 
fermentation  into  one  that  does. 

Coagulating,  fat-splitting,  hydrolytic  ferments  are  some  of  the 
other  varieties. 

Toxins  and  Toxalbumins  are  various  albuminoids  produced 
in  the  animal  organism  and  in  culture-media  which  are  very 
poisonous,  and  are  considered  the  prime  cause  of  disease. 

Pigmentation.  Some  bacteria  are  endowed  with  the  property 
of  forming  pigments  either  in  themselves,  or  producing  a  thro- 
mogenic  body  which,  when  set  free,  gives  rise  to  the  pigment. 
In  some  cases  the  pigments  have  been  isolated  and  many  of  the 
properties  of  the  aniline  dyes  discovered  in  them. 

Phosphorescence.     Many  bacteria  have  the  power  to  form 


Zb         ESSENTIALS  OF  BACTRIOLOGY. 

light,  giving  to  various  objects  which  they  inhabit  a  character- 
istic glow  or  phosphorescence. 

Fluorescence.  An  iridescence,  or  play  of  colors,  develops  in 
some  of  the  bacterial  cultures. 

Gas  Formation.  Many  bacteria,  anaerobic  ones  especially, 
produce  gases,  noxious  and  odorless  ;  in  the  culture-media  the 
bubbles  which  arise  soon  displace  the  media. 

Odors.  Some  germs  form  odors  characteristic  of  them:  some 
are  pleasant  and  even  fragrant;  others,  foul  and  nauseous. 

Effect  of  Age.     With  age,  bacteria  lose  their  strength  and  die. 


CHAPTER  III. 

METHODS   OF   EXAMINATION. 

We  divide  the  further  study  of  the  general  characteristics  of 
Bacteria  into  two  portions  : — 

First.    The  examination  of  bacteria  by  aid  of  the  microscope. 

Second.    The  continued  study  through  artificial  cultivation. 

They  both  go  hand  in  hand  ;  the  one  incomplete  without  the 
other. 

Microscopical.  The  ordinary  microscope  will  not  suffice  for 
Bacteriologic  research.  Certain  special  appliances  must  first 
be  added.  It  is  not  so  much  required  to  have  a  picture  very 
large,  as  to  have  it  sharp  and  clear. 

Oil  Immersion  Lens.  The  penetration  and  clearness  of  a  lens 
are  very  much  influenced  by  the  absorption  of  the  rays  of  light 
emerging  from  the  picture.  In  the  ordinary  dry  system,  many 
of  the  light  rays,  being  bent  outward  by  the  air  which  is  be- 
tween the  object  and  the  lens,  do  not  enter  the  lens,  and  are 
lost.  By  interposing  an  agent  which  has  the  same  refractive 
index  as  glass,  cedar-oil,  or  clove-oil,  for  example,  all  the  rays 
of  light  from  the  object  enter  directly  into  the  lens. 

The  "  Homogeneous  System/'  or  oil-immersion  lens,  consists 
of  a  system  of  lenses  which  can  be  dipped  into  a  drop  of  cedar- 
oil  placed  upon  the  cover-glass,  and  which  is  then  ready  for  use. 


METHODS    OF    EXAMINATION. 


27 


Abbe's  Condenser.     The  second  necessary  adjunct  is  a  com- 
bination of  lenses  placed  underneath  the  stage,  for  bringing 
wide  rays  of  light   directly  under 
the  object.     It  serves   to  intensify  Fig.  7. 

the   colored   pictures  by  absorbing 
or  hiding  the  unstained  structure. 

This  is  very  useful  in  searching 
a  specimen  for  bacteria,  since  it 
clears  the  field  of  everything  that 
is  not  stained.  It  is  called  Abbe's 
Condenser.       Together    With    it    is  Abbe's  Condenser. 

usually   found  an    instrument  for 

shutting  off  part  of  the  light — a  blender  or  diaphragm.  When 
the  bacteria  have  been  found,  and  their  relation  to  the  structure 
is  to  be  studied,  the  "  Abbe* "  is  generally  shut  out  by  the  iris 
blender,  and  the  structure  comes  more  plainly  into  view.  A 
white  light  (daylight  or  a  Welsbach  burner)  is  best  for  bacterial 
study :  use  the  plane  mirror  for  daylight  and  the  concave 
mirror  for  artificial  light. 


Iris  Blender. 


For  all  stained  Bacteria  the  oil  immersion  lens  and  Abbe  con- 
denser, without  the  use  of  blender.  For  unstained  specimens, 
oil  immersion  and  the  narrowed  blender. 

When  examining  with  low  power  objective,  use  a  strong 
ocular.  When  using  high  power  objective  use  weak  ocular.  A 
nose-piece  will  be  found  very  useful,  since  it  is  sometimes  neces- 


28         ESSENTIALS  OF  BACTERIOLOGY. 

sary  to  change  the  objective  on  the  same  field,  and  this  insures 
a  great  steadiness  of  the  object. 

Great  cleanliness  is  needed  in  all  bacteriological  methods  ;  but 
nowhere  more  so  than  in  the  microscopical  examination. 

The  cover-glass  should  be  very  carefully  washed  in  alcohol, 
and  dried  with  a  soft  linen  rag.  To  remove  the  stains  on  the 
cover-glasses  that  have  been  used,  they  should  be  soaked  in 
hydrochloric  acid  or  placed  in  a  6  per  cent,  aqueous  solution  of 
potassium  bichromate  with  6  per  cent,  of  strong  sulphuric  acid, 
washed  in  water,  and  kept  in  absolute  alcohol. 

Examination  of  Unstained  Bacteria.    As  the  coloring  of  bac- 

Fig.  9. 


Platinum  Needles. 

teria  kills  them  and  changes  their  shape  to  some  extent,  it  is  pre- 
ferable to  examine  them  when  possible  in  their  natural  state. 

We  obtain  the  bacteria  for  examination,  either  from  liquid  or 
solid  media. 

From  Liquids.  With  a  long  platinum  needle,  the  end  of  which 
is  bent  into  a  loop,  we  obtain  a  small  drop  from  the  liquid  con- 
taining the  bacteria,  and  place  it  on  a  cover-glass  or  slide  ; 
careful  that  no  bubbles  remain. 

Sterilize  Instruments.  Right  here  we  might  say  that  it  is 
best  to  accustom  one's  self  to  passing  all  instruments,  needles, 
etc.,  through  the  flame  before  and  after  each  procedure  ;  it  in- 
sures safety  ;  and  once  in  the  habit,  it  will  be  done  automati- 
cally. 

From  Solid  Media.  With  a  straight-pointed  platinum  needle, 
a  small  speck  of  the  medium  is  taken  and  rubbed  upon  a  glass 


METHODS    OF    EXAMINATION 


29 


slide,  with  a  drop  of  sterilized  water,  or  bouillon,  and  from  this 
a  little  is  taken  on  cover-glass,  as  before. 

The  cover-glass  with  its  drop  is  now  placed  on  the  glass  slide, 
carefully  pressing  out  all  bubbles.  Then  a  drop  of  cedar-oil  is 
laid  on  top  of  the  cover-glass,  and  the  oil  immersion  lens  dipped 
gently  down  into  it  as  close  as  possible  to  the  cover-glass, 
the  narrow  blender  shutting  off  the  Abbe  condenser,  for  this 
being  an  unstained  specimen,  we  want  but  little  light  We 
now  apply  the  eye,  and  if  not  in  focus,  use  the  fine  adjust- 
ment or  the  coarse,  but  always  away  from  the  object — i.  e.  towards 
us  —  since  the  distance  between  the  specimen  and  the  lens 
is  very  slight,  it  does  not  require  much  turning  to  break  the 
cover-glass  and  ruin  the  specimen.  Having  found  the  bacte- 
rium, we  see  whether  it  be  bacillus,  micrococcus,  or  spirillum  ; 
discover  if  it  be  motile,  or  not.  That  is  about  all  we  can  ascer- 
tain by  this  method. 

Fig.  10. 


Hanging  Drop  in  Concave  Glass  Slide. 


Hanging  Drop.  When  the  looped  platinum  needle  is  dipped 
into  a  liquid,  a  very  finely-formed  globule  will  hang  to  it ;  this 
can  be  brought  into  a  little  cupped  glass  slide  (an  ordinary 
microscopic  glass  slide  with  a  circular  depression  in  the  centre) 
in  the  following  manner :  The  drop  is  first  brought  upon  a 
cover-glass ;  the  edges  of  the  concavity  on  the  glass  slide  are 
smeared  with  vaseline,  and  the  slide  inverted  over  the  drop; 
the  cover-glass  sticks  to  the  smeared  slide,  which,  when  turned 


30  ESSENTIALS    OF    BACTERIOLOGY. 

over,  holds  the  drop  in  the  depression  covered  by  the  cover-glass, 
thus  forming  an  air-tight  cell;  here  the  drop  cannot  evaporate. 
Both  slide  and  cover-glass  should  first  be  sterilized  by  heat. 

Search  for  the  bacteria  with  a  weak  lens  ;  having  found  them, 
place  a  drop  of  cedar-oil  upon  the  cover-glass,  and  bring  the  oil 
immersion  into  place  (here  is  where  a  nose-piece  comes  in  very 
usefully),  careful  not  to  press  against  the  cell,  for  the  cover- 
glasses  are  very  fragile  in  this  position. 

Search  the  edges  of  the  drop  rather  than  the  middle  ;  the  bac- 
teria will  usually  be  very  thick  in  the  centre  and  not  so  easily 
distinguished. 

Spores,  automatic  movements,  fission,  and  cultivation  in 
general  can  be  studied  for  several  days.  This  moist  chamber 
can  be  placed  in  a  brood-oven  or  on  the  ordinary  warming 
stages  of  the  microscope. 

Agglutination  as  observed  in  Widal's  test  is  best  seen  in  the 
hanging  drop. 


CHAPTER  IV. 

STAINING    OF   BACTERIA. 

Staining  or  coloring  bacteria  is  done  in  order  to  make  them 
prominent,  and  to  obtain  permanent  specimens.  It  is  also 
necessary  to  bring  out  the  structure  of  the  bacteria,  and 
serves  in  many  instances  as  a  means  of  diagnosis  ;  and  lastly, 
it  would  be  well-nigh  impossible  to  discover  them  in  the  tissues, 
without  staining. 

Anilin  Colors.  Of  the  numerous  dyes  in  the  market,  nearly 
all  have,  at  one  time  or  other,  been  used  in  staining  bacteria. 
But  now  only  a  very  few  find  general  use,  and  with  methyline 
blue  and  fuchsin  nearly  every  object  can  be  accomplished. 

Basic  and  Acid  Dyes.    Ehrlich  was  the  first  to  divide  the 
anilin  dyes  into  two  groups,  the  basic  colors  to  which  belong — 
Gentian  violet,  or  pyoktanin,  Basic  fuchsin. 

Methyl  violet,  or  dahlia,  Bismarck-brown, 

Methylin  blue  {not  methyl  blue),  Thionin, 

Saffranin. 
And  the  acid  colors  to  which  eosin  and  acid-fuchsin  belong. 


STAINING    OF    BACTERIA.  31 

The  basic  dyes  stain  the  bacteria  and  the  nuclei  of  cells  ;  the 
acidE  dyes  stain  chiefly  the  tissue,  leaving  the  bacteria  almost 
untouched.  Carmine  and  Hematoxylin  are  also  useful  as  con- 
trast stains,  affecting  bacteria  very  slightly.  The  anilin  dyes 
are  soluble  in  alcohol  or  water  or  a  mixture  of  the  two. 

Staining  Solutions.  A  saturated  solution  of  the  dye  is  made 
with  alcohol.  This  is  called  the  stock  or  concentrated  solution ; 
1  part  of  this  solution  to  about  100  parts  of  distilled  water  con- 
stitutes the  ordinary  aqueous  solution  in  use  or  weak  solution. 

It  is  readily  made  by  adding  to  an  ounce  bottle  of  distilled 
water  enough  of  the  strong  solution  until  the  fluid  is  still  opaque 
in  the  body  of  the  bottle,  but  clear  in  the  neck  of  the  same. 

These  weak  solutions  should  be  renewed  every  three  or  four 
weeks,  otherwise  the  precipitates  formed  will  interfere  with  the 
staining. 

Compound  Solutions.  By  means  of  certain  chemical  agents, 
the  intensity  of  the  aniline  dyes  can  be  greatly  increased. 

Mordants.  Agents  that  t(p  6iie"  into  the  specimen  carrying 
the  stain  with  them,  depositing  it  in  the  deeper  layers,  are 
called  mordants  or  etchers. 

Various  metallic  salts  and  vegetable  acids  are  used  for  such 
purpose. 

The  mother  liquid  of  the  anilin  dyes,  anilin  oil,  a  member  of 
the  aromatic  benzol  group,  has  also  this  property. 

Anilin  Oil  Water.  Anilin  oil  is  shaken  up  with  water  and 
then  filtered;  the  anilin  water  so  obtained  is  mixed  with  the 
dyes,  forming  the  "anilin  water  gentian  violet"  or  anilin  water 
fuchsin,  etc. 

Carbol  Fuchsin.  Carbolic  acid  can  be  used  instead  of  anilin 
oil,  and  forms  one  of  the  main  ingredients  of  Ziehl's  or  Neelsen's 
solution,  used  principally  in  staining  bacillus  tuberculosis. 
Kuhne  has  a  carbol- methylin  blue  made  similar  to  the  carbol 
fuchsin. 

Alkaline  Stains.  Alkalies  have  the  same  object  as  the  above 
agents ;  namely,  to  intensify  the  picture.  Potassium  hydrate, 
amnion,  carbonate,  and  sodium  hydrate  are  used. 

Loffier's  alkaline  blue  and  Koch's  weak  alkaline  blue  have  in 
them  potassium. 


32  ESSENTIALS   OP    BACTERIOLOGY. 

Heat.  Warming  or  boiling  the  stains  during  the  process  of 
staining  increases  their  intensity. 

Decolorizing  Agents.  The  object  is  usually  over-colored  in 
some  part,  and  then  decolorizing  agents  are  employed.  Water  is 
sufficient  for  many  cases  ;  alcohol  and  strong  mineral  acids  com- 
bined are  necessary  in  some. 

Iodin  as  used  in  Gram's  Method.  Belonging  to  this  group, 
but  used  more  in  the  sense  of  a  protective,  is  tincture  of 'iodin. 
It  picks  out  certain  bacteria,  which  it  coats;  prevents  them  from 
being  decolorized,  but  allows  all  else  to  be  faded.  Then  by 
using  one  of  the  acid  or  tissue  dyes,  a  contrast  color  or  double 
staining  is  obtained.  Many  of  the  more  important  bacteria  are 
not  acted  upon  by  the  iodin,  and  it  thus  becomes  a  very  useful 
means  of  diagnosis. 

Formulas  of  different  Staining  Solutions. 

I. — Saturated  Alcoholic  Solution. 
Place  about  10  grammes  of  the  powdered  dye  in  a  bottle  and 
add  40  grammes  of  alcohol.      Shake  well  and  allow  to  settle. 
This  can  be  used  as  the  stock  bottle. 

II. —  Weak  Solutions. 
Made  best  by  adding  about  1  part  of  number  I.  or  stock  solu- 
tion to  10  of  distilled  water.    This  is  the  ordinary  solution  in  use. 

III. — Anilin  Oil  Water. 

Aniline  oil 5  parts. 

Distilled  water        .        .  *      .         .     100  parts.— M. 
Shake  wTell  and  filter.     To  be  made  fresh  each  time. 

IV.— Anilin  Water  Dyes. 
Sat.  alcoh.  sol.  of  the  dye       .         .       11  parts. 
Aniline  oil  water    ....     100  parts. 

Abs.  alcohol 10  parts. — M. 

Can  be  kept  10  days. 


STAINING    OF    BACTERIA.  33 

V.—  Alkaline  Methylin  Blue. 

A.  Loffler's. 

Sat.  ale.  Sol.  methylin  blue     .         .       30 
Sol.  potass,  hydrat.  (1-10,000)         .     100— M. 

B.  Koch's. 

Sol.  potass,  hydrat.  (10  per  eent.)  0.2 

Sat.  ale.  sol.  methyl,  blue       .         .  1.0 

•       Distilled  water        ....  200.0— M. 

VI. —  Carbolic  Acid  Solutions. 

A.  Ziehl-Neelsen. 

Fuehsin  (powd.)      ....  1  part. 

Alcohol 10  parts. 

5  per  eent.  sol.  acid,  carbolic          .  100  parts.— M. 
Filter.     The  older  the  solution  the  better. 

B.  Kilhne. 

Methylin  blue  .         .        .         .         1.5 

Alcohol 10.0 

5  per  cent.  sol.  ac.  carbol.       .        .     100.0 
Add  the  acid  gradually.     This  solution  loses  strength  with  age. 

VII. — Gram's  Iodin  Solution. 

Iodine 1 

Potass,  iod 2 

Aquae  destillat 300.— M. 

VIII.— Loffler's  Mordant  (for  flagella). 
Aq.  sol.  of  tannin  (20  per  cent.)     .       10  parts. 
Aq.  sol.  ferri  sulph.  (5  per  cent.)    .         1  part. 
Aqua?  decoc.  of  logwood  (1-8)        .        4  parts. — M. 
Keep  in  well-corked  bottle. 

IX. —  Unna's  Borax  Methyl  Blue. 

Borax 1  part. 

Methyl  blue 1  part. 

Water 100  parts.— M. 


■6 


34         ESSENTIALS  OF  BACTERIOLOGY. 

X. — Gobbet's  Acid  Blue  (rapid  stain). 
Methylin  blue  ....        2 

25  per  cent,  sulphuric  acid      .        .     100.— M. 

XI. — Alkaline  Anilin   Water  Solutions. 
Sodium  hydrat.  (1  per  cent.)  .        .        1 
Anilin  oil  water      ....     100. — M. 
And  add — 

Fuchsin,  or  methyl-violet  powd.    .        4 
Cork  well.     Filter  before  using. 

XII. — Roux's  Double  Stain. 
Dahlia  or  gentian  violet  ...        0.5  gramme. 

Methyl  green 1.5        " 

Distilled  water 200.0 grammes. — M. 

Use  as  other  stains,  without  acid. 


XIII. — Neisser's  Stain.     (For 

Diphtheria.) 

Solution  I. 

Methylin  blue  .... 
Alcohol  (96  per  cent.) 
Dissolve  and  add  water    . 
Glacial  acetic  acid    . 

1  gramme. 
.      20  c.c. 
.     950  c.c. 
.      50  c.c— M. 

Solution  II. 

Vesuvin 

Water 

2  grammes. 
.  1000  c.c— M. 

Stain  cover-glasses  (1)  three  seconds  in  Sol.  I. ;  (2)  wash  in 
water;  (3)  three  seconds  in  No.  2;  (4)  wash  in  water.  Body  of 
bacillus,  brown ;  oval  granules  at  each  end,  blue. 

XIV. — Carbol-thionin.     (Nicolle.) 

Sat.  sol.  thionin  in  ale  (90  per  cent.)       10  c.c. 
Aqueous  sol.  ac  carbol.  (1  per  cent.)  100  c.c. — M. 
Stain  sections,  one-half  to  one  minute. 


METHOD    OF    STAINING    SPECIMENS.  35 

XV. — Capsule  Stain  of  Hiss. 
Use  the  following,  heated  until  it  steams : 

Sat.  alcoholic  solution  of  1  K  „  „ 

l      .  o  c.c. 

gentian  violet  or  fuchsin  j 

Distilled  water  ....      95  c.c. 

Wash  in  20  per  cent,  solution  of  cupric  sulphate  crystals. 

XVI. — Capsule  Stain  of  Welch. 
(1)  Pour  glacial  acetic  acid  on  film.  After  a  few  seconds 
replace  with  anilin-water  gentian  violet  without  washing  in 
water.  (2)  Remove  all  acid  by  several  additions  of  stain,  and 
allow  it  to  act  for  three  to  four  minutes.  (3)  Wash  and  ex- 
amine in  salt  solution  0.8-2.0  per  cent. 


CHAPTER  V. 

GENERAL   METHOD   OF    STAINING   SPECIMENS. 

Cover-Glass  Preparations.  The  material  is  evenly  spread  in 
as  thin  a  layer  as  possible  upon  a  cover-glass  ;  then,  to  spread 
it  still  more  finely,  a  second  cover-glass  is  pressed  down  upon 
the  first  and  the  two  slid  apart.  This  also  secures  two  speci- 
mens. Before  they  can  be  stained  they  must  be  perfectly  dry, 
otherwise  deformities  will  arise  in  the  structure. 

Drying  the  Specimen.— The  cover-glass  can  be  set  aside  to  dry, 
or  held  in  the  fingers  over  the  Bunsen  burner  (the  fingers  prevent- 
ing too  great  a  degree  of  heat).  Since  most  of  the  specimens 
contain  a  certain  amount  of  albumenoid  material,  it  is  best  in 
all  cases  to  "fix"  it,  t,  e.,  to  coagulate  the  albumen.  This  is 
accomplished  by  passing  the  cover-glass  (after  the  specimen  is 
dry)  three  times  through  the  flame  of  the  burner,  about  three 
seconds  being  consumed  in  doing  so,  the  glass  being  held  in  a 
small  forceps,  smeared  side  up. 

The  best  forceps  for  grasping  cover-glasses  is  a  bent  one,  bent 
again  upward,  near  the  ends.  (Fig.  11.)  It  prevents  the  flame 
or  staining-fluid  from  reaching  the  fingers. 


36  ESSENTIALS    OF    BACTERIOLOGY. 

The  object  is  now  ready  for,  staining. 

Staining.— A  few  drops  of  the  staining  solution  are  placed 
upon  the  cover-glass  so  that  the  whole  specimen  is  covered, 
and  it  is  left  on  a  few  minutes,  the  time  depending  upon  the 
variety,  the  strength  of  stain,  and  the  object  desired.     Instead 

Fig.  11. 


Author's  Bent  Forceps  for  Holding  Cover-glass  over  Flame. 

of  placing  the  dye  upon  the  object,  the  cover-glass  can  be  im- 
mersed in  a  small  glass  dish  containing  the  solution  ;  or,  if 
heat  is  desired  to  intensify  or  hasten  the  process,  a  watch- 
crystal  holding  the  stain  is  placed  over  a  Bunsen  burner  and 
in  it  the  cover-glass  ;  and,  again,  the  cover-glass  can  be  held 
directly  in  the  flame  with  the  staining  fluid  upon  it,  which 
must  be  constantly  renewed  until  the  process  is  completed, 
or  the  cover-glass  can  be  heated  in  a  test-tube. 

Removing  Excess  of  Stain.  The  surplus  stain  is  washed  off 
by  dipping  the  glass  in  distilled  water. 

The  water  is  removed  by  drying  between  filter  paper  or 
simply  allowed  to  run  off  by  standing  the  cover-glass  slant- 
wise against  an  object.  When  the  specimen  is  to  be  examined 
in  water  (which  is  always  best  with  the  first  preparation  of 
the  specimen,  as  the  Canada  balsam  destroys  to  some  extent 
the  natural  appearance  of  the  bacteria),  a  small  drop  of  ster- 
ilized water  is  placed  upon  the  glass  slide,  and  the  cover-glass 
dropped  gently  down  upon  it,  so  that  the  cover-glass  remains 
adherent  to  the  slide. 

The  dry  system  or  the  oil-immersion  can  now  be  used. 

When  the  object  has  been  sufficiently  examined  it  can  be  per- 
manently mounted  by  lifting  the  cover-glass  off  the  slide  (this 
is  facilitated  by  letting  a  little  water  flow  under  it,  one  end 


METHOD    OF    STAINING    SPECIMENS.  37 

being  slightly  elevated).  The  water  that  still  adheres  is  dried 
off  in  the  air  or  gently  over  the  flame,  and  when  perfectly  dry 
it  is  placed  upon  the  drop  of  Canada  balsam  which  has  been  put 
upon  the  glass  slide. 

In  placing  the  cover- glass  in  the  staining  solutions  one  must 
be  careful  to  remember  which  is  the  spread  side. 

By  holding  it  between  yourself  and  the  window,  and  scraping 
the  sides  carefully  with  the  sharp  point  of  the  forceps,  the  side 
having  the  specimen  on  it  will  show  the  marks  of  the  instrument. 

Little  glass  dishes,  about  one-half-dozen,  should  be  at  hand 
for  containing  the  various  stains  and  decolorants. 

Tissue  Preparations.  In  order  to  obtain  suitable  specimens 
for  staining,  very  thin  sections  of  the  tissue  must  be  made. 

As  with  histological  preparations,  the  tissue  must  be  hardened 
before  it  can  be  cut  thin  enough.  Alcohol  is  the  best  agent  for 
this  purpose. 

Fig.  12. 


I 


'         : 


Spatula  for  Lifting  Sections. 

Pieces  of  the  tissue  one-quarter  inch  in  size  are  covered  with 
alcohol  for  24  to  48  hours. 

When  hardened  it  must  be  fixed  upon  or  in  some  firm  object. 
A  paste  composed  of— 

Gelatine 1  part. 

Glycerine 4  parts. 

Water 2  parts. 

will  make  it  adhere  firmly  to  a  cork  in  about  2  hours,  or  it  can 
be  imbedded  in  a  small  block  of  paraffine,  and  covered  over  with 
melted  paraffine.  Celloidin  may  be  used  as  an  imbedding  agent, 
and  formalin  is  useful  to  harden  tissue  quickly. 

Cutting.  The  microtome  should  be  able  to  cut  sections  j^B 
inch  in  thickness  ;  this  is  the  fineness  usually  required. 

The  sections  are  brought  into  alcohol  as  soon  as  cut  unless 
they  have  been  imbedded  in  paraffine,  when  they  are  first  washed 
in  chloroform  to  dissolve  out  the  paraffine. 


38 


ESSENTIALS  OF  BACTERIOLOGY. 


Staining.  All  the  various  solutions  should  be  in  readiness, 
best  placed  in  the  little  dishes  in  the  order  in  which  they  are  to 
be  used,  as  a  short  delay  in  one  of  the  steps  may  spoil  the  speci- 
men. 

A  very  useful  instrument  for  transferring  the  delicate  sections 
from  one  solution  to  another  is  a  little  metal  spatula,  the  blade 
being  flexible. 

A  still  better  plan,  especially  when  the  tissue  is  "crumbling/ 
is  to  carry  out  the  whole  procedure  on  the  glass-slide. 

Fig.  13. 


Section  Microtome. 


General  Principles.  The  section  is  transferred  from  the  alco- 
hol in  which  it  has  been  kept  into  water,  which  removes  the 
excess  of  alcohol,  from  here  into — 

Dish  I,  containing  the  stain;  where  it  remains  5  to  15  minutes. 
Then- 

JJish  II,  containing  5  per  cent,  acetic  acid  (1  to  20);  where  it 
remains  ^  to  1  min.     The  acid  removes  the  excess  of  stain. 

Dish  III,  water  to  rinse  off  the  acid.  The  section  can  now  be 
placed  under  the  microscope  covered  with  cover-glass  to  see  if 
the  intensity  of  the  stain  is  sufficient  or  too  great.     A  second 


STAINING   AND    MODIFICATIONS.  39 

section  is  then  taken,  avoiding  the  errors,  if  any ;  and  having 
reached  this  stage  proceeded  with  as  follows  : — 

Dish  IV,  alcohol,  2  to  3  seconds  to  remove  the  water  in  the 
tissue. 

V.  A  few  drops  of  oil  of  cloves,  just  long  enough  to  clear  the 
specimen  to  make  it  transparent  (so  that  an  object  placed  under 
neath  will  shine  through). 

VI.  Remove  excess  with  filter-paper. 

VII.  Mount  in  Canada  balsam  (xylol  balsam). 


CHAPTER  VI. 

SPECIAL  METHODS  OF  STAINING  AND  MODIFICATIONS. 

Gram's  Method  of  Double  Staining.  (For  cover-glass  speci- 
mens.)— I.  A  hot  solution  of  anil,  water  gentian  violet  2  to  10 
minutes. 

II.  Directly  without  washing,  into  Gram's  solution  of  iod. 
potass,  iod.  1  to  3  min.  (the  cover-glass  looks  black). 

III.  Wash  in  alcohol  60  per  cent,  until  only  a  light  brown 
shade  remains  (as  if  the  glass  were  smeared  with  dried  blood). 

IV.  Rinse  off  alcohol  with  water. 

V.  Contrast  color  with  either  eosin,  picro-carmine,  or  bismark- 
brown.  The  bacteria  will  appear  deep  blue,  all  else  red  or  brown 
on  a  very  faint  brown  background. 

The  following  bacteria  do  not  retain  their  color  with  Gram's 
method — are  therefore  not  available  for  the  stain :  Bacillus  of 
typhoid ;  spirillum  of  cholera ;  bacillus  of  chicken  cholera,  of 
hemorrhagic  septicaemia,  of  malignant  oedema,  of  pneumonia 
(Friedlander),  and  of  glanders;  diplococcus  of  gonorrhoea ;  spi- 
rillum of  relapsing  fever. 

Gram's  Method  for  Tissues  (modified  by  Giinther). 

I.  Stain  in  anil,  water  gent,  violet      .        .    1  minute. 
II.  Dry  between  filter  paper. 

III.  Iod.  potass,  iod.  sol.         ....    2  minutes. 

IV.  Alcohol £  minute. 

V.  3  perct.  sol.  hydrochloric  acid  in  alcohol     10  seconds. 

VI.  Alcohol,  ol.  of  cloves,  and  Canada  balsam. 


40  ESSENTIALS    OF    BACTERIOLOGY. 

To  Stain  Spores.  Since  spores  have  a  very  firm  capsule, 
which  tends  to  keep  out  all  external  agents,  a  very  intensive 
stain  is  required  to  penetrate  them,  but  once  this  object  attained 
it  is  equally  as  difficult  to  decolorize  them. 

A  cover-glass  prepared  in  the  usual  way,  i.  e.,  drying  and 
passing  the  specimen  through  the  flame  three  times,  is  placed  in 
a  watch-crystal  containing  Ziehl's  carbol-fuchsin  solution,  and 
the  same  placed  upon  a  rack  over  a  Bunsen  burner,  where  it  is 
kept  at  boiling-point  for  one  hour,  careful  to  supply  fresh  solution 
at  short  intervals  lest  it  dry  up. 

The  bacilli  are  now  decolorized  in  alcohol,  containing  £  per 
cent,  hydrochloric  acid.  A  contrast  color,  preferably  methylin 
blue,  is  added  for  a  few  minutes. 

The  spores  will  appear  as  little  red  beads  in  the  blue  bacteria, 
and  loose  ones  lying  about. 

Spore  Stain  (modified). — I.  Carbol-fuchsin  on  cover-glass  and 
heated  in  the  flame  to  boiling  point  20  to  30  times. 

II.  25  per  cent,  sulphuric  acid,  2  seconds  ;  rinsed  in  water. 

III.  Methylin  blue  contrast. 

Alex.  Klein  recommends  the  following  spore  method  :  mix  a 
little  of  the  culture  (potato)  with  3  drops  of  physiologic  salt 
solution,  and  heat  gently  with  an  equal  quantity  of  carbol- 
fuchsin  for  a  period  of  6  minutes.  Spread  then  on  cover-glasses, 
dry  in  the  air,  and  fix  by  passing  three  times  through  Bunsen 
burner  flame.  Decolorize  in  1  per  cent,  sulphuric  acid  for  1  to 
2  seconds ;  contrast  in  weak  methylin  blue. 

BowhilVs  Orcein  Stain. 
Sat.  alcoholic  solution  of  orcein  .        .        .15  c.c. 
20  per  cent,  aqueous  sol.  tannin  .        .        .10  c.c. 

Distilled  water 30  c.c. — M. 

Filter. 

Use  orcein  solution  in  watch-glass,  float  cover-glass  in  it  and 
heat  gently,  not  boil,  for  10  minutes.  Wash  in  water.  Dry  and 
mount  in  balsam. 

Five  per  cent,  chromic  acid  applied  for  15  minutes  has  been 
recommended  in  staining  spores.  This  is  followed  by  the 
carbol-fuchsin  stain  as  above. 


STAINING   AND    MODIFICATIONS.  41 

Flagella  Stain,  with  Loffier's  Mordant. — I.  A  few  drops  of  the 
mordant  (No.  viii.  p.  33)  are  placed  upon  the  spread  cover-glass 
and  heated  until  it  steams. 

II.  Washed  with  water  until  the  cover-glass  looks  almost  clean, 
using  a  small  piece  of  filter  paper  to  rub  off  the  crusts  which  have: 
gathered  around  the  edges. 

III.  Anilin  water  fuchsin  (neutral)  held  in  flame  about  1£ 
minutes. 

IV.  Wash  in  water. 

If  the  stain  is  properly  made,  the  microbes  are  deeply  colored 
and  the  flagella  seen  as  little  dark  lines  attached  to  them. 

Sporogenic  bodies  stain  quite  readily,  and  in  order  to  distin- 
guish them  from  spores  Ernst  uses  alkaline  methylin  blue,  slightly 
warmed.  Then  rinse  in  water.  Contrast  with  cold  bfemark- 
brown. 

The  spores  are  colored  bright  blue,  the  spore  granules  a  dirty 
blue,  being  mixed  with  the  brown,  which  colors  also  the  bacteria. 

Kiihne's  Method. — In  sections,  the  alcohol  used  sometimes  de- 
colorizes too  much.  To  obviate  this  Kulme  mixes  the  alcohol 
with  the  stain,  so  that  while  the  section  is  being  anhydrated  it 
is  constantly  supplied  with  fresh  dye. 

Weigert  uses  aniline  oil  to  dehydrate  instead  of  alcohol,  and 
here,  too,  it  can  be  used  mixed  with  the  dye. 

Unna's  Method  for  Fungi  (especially  useful  for  epidermic; 
scales). — Moisten  horny  scale  or  crust  with  acetic  acid;  mace- 
rate between  two  glass  slides ;  dry  in  flame ;  wash  out  fat  with 
ether  and  alcohol  (equal  parts) ;  stain  in  borax  methyl  blue  for 
ten  seconds  (over  flame) ;  bleach  with  glycerine  and  aether  (equal 
parts) ;  rinse  in  water,  alcohol,  dry,  and  mount. 

Behavior  of  the  More  Important  Bacteria  to  Gram's  Stain. 

Positive  means  that  the  bacteria  retain  the  primary  color,  or 
gentian  violet. 

Positive.  Negative. 

Tubercle  Bacillus,  Colon  Bacillus, 

Smegma  Bacillus,  Typhoid  Bacillus, 

Lepra  Bacillus,  Cholera  Bacillus, 

Anthrax  Bacillus,  Influenza  Bacillus, 


42                      ESSENTIALS  OF   BACTERIOLOGY. 

Positive.  Negative. 

Tetanus  Bacillus,  Friedlander's  Bacillus, 

Diphtheria  Bacillus,  Plague  Bacillus, 

Pneumococcus,  Diplococcus  intracellularis, 

Streptococcus,  Gonococcus, 

Staphylococcus,  Koch-Weeks'  Bacillus, 

Cocci  of  the  urethra.  Conjunctivitis  Bacillus  of  Moras. 


CHAPTER  VII. 

METHODS   OF   CULTURE. 

Artificial  Cultivation.— The  objects  of  cultivation  are  to  obtain 
germs  in  pure  culture,  free  from  all  foreign  matter,  isolated  and 
so  developed  as  to  be  readily  used  either  for  microscopical  ex- 
amination or  animal  experimentation. 

To  properly  develop  bacteria  we  supply  as  near  as  possible 
the  conditions  which  hold  for  the  especial  germ  in  nature. 
With  the  aid  of  solid  nutrient  media  the  bacteria  can  be  easily 
separated,  and  the  methods  are  nearly  perfect. 

Sterilization.  If  we  place  our  nutrient  material  in  vessels 
that  have  not  been  properly  disinfected,  we  will  obtain  growths 
of  bacteria  without  having  sown  any. 

If  we  have  thoroughly  cleaned  our  utensils,  and  then  not  taken 
care  to  protect  them  from  further  exposure,  the  germs  we  have 
sown  will  be  effaced  or  contaminated  by  multitudes  of  others, 
that  are  constantly  about  us.  We  therefore  have  two  neces- 
sary precautions  to  take  : — 

First.  To  thoroughly  clean  and  sterilize  every  object  that 
enters  into,  or  in  any  way  comes  in  contact  with,  the  culture. 

Second.  To  maintain  this  degree  of  sterility  throughout 
the  whole  course  of  the  growth,  and  prevent,  by  proper  con- 
tainers, the  entrance  of  foreign  germs. 

Disinfectants.  Corrosive  sublimate  (bichloride  of  mercury), 
which  is  the  most  effective  agent  we  possess,  cannot  be  gene- 
rally used  because  it  renders  the  soil  unproductive  and  therefore 


METHODS    OF    CULTURE. 


43 


must  only  be  employed  in  washing  dishes,  to  destroy  the  old 
cultures.  Even  after  washing,  a  few  drops  of  the  solution  may 
remain  and  prevent  growth,  so  that  one  must  be  careful  to  have 
the  glass-ware  that  comes  in  contact  with  the  nutrient  media 
free  from  the  sublimate. 

Heat.  Heat  is  the  best  agent  we  possess  for  general  use. 
Dry  heat  and  moist  heat  are  the  two  forms  employed,  but  these 
differ  greatly  in  effectiveness.  Thus  Koch  found  that  while 
moist  heat  at  100°  C.  killed  the  spores  of  the  anthrax  bacillus 
in  one  hour,  it  required  three  hours  of  dry  heat  at  140°  C.  to 
produce  death. 

Fig.  14. 


Hot  Air  Oven. 

For  obtaining  dry  heat— that  is,  a  temperature  of  150°  C, 
(about  300°  F.)— a  sheet-iron  oven  is  used  which  can  be  heated 
by  a  gas-burner.  If  it  have  double  walls  (air  circulating  be- 
tween), the  desired  temperature  is  much  more  quickly  obtained. 
A  small  opening  in  the  top  to  admit  a  thermometer  is  neces- 
sary. These  chests  are  usually  about  1  foot  high,  1^  foot  wide, 
and  f  foot  deep.  In  them,  glassware,  cotton,  and  paper  can  be 
sterilized.     "When  the  cotton  is  turned  slightly  brown,  it  usually 


44 


ISSENTIALS    OF   BACTERIOLOGY, 


denotes  sufficient  sterilization.  All  instruments,  where  practi- 
cable, should  be  drawn  through  the  flame  of  an  alcohol  lamp  or 
Bunsen  burner.  One  hour  in  the  oven  at  170°  C.  usually  steri- 
lizes glass-ware,  while  the  ordinary  germs  in  liquids  may  be 

Fig.  15. 


Koch's  Steam-chest. 


killed  by  boiling  for  five  minutes  if  no  spores  are  present.  The 
boiling  of  any  fluid  at  100°  C.  for  one  and  one-half  hours  nearly 
always  ensures  sterilization. 

Moist  Heat. — Steam  at  100°  C.  in  circulation  has  been  shown 
to  be  a  very  effective  application  of  heat. 


METHODS    OF    CULTURE. 


45 


Koch's  Steam-chest.  Circulating  steam  is  obtained  by  aid  of 
Koch's  apparatus.  This  consists,  of  a  cylindrical  tin  chest 
.about  2£  feet  high  and  about  £  foofom  diameter  ;  divided  in  its 
interior  by  a  perforated  diaphragtiJja,  an  upper  chamber  for 
the  steam,  c,  and  a  lower  one  for ,, water,  b.  Two  or  more 
gas-burners  placed  underneath  the  chest,  which  stands  on   a 


Fig.  16. 


Arnold's  Steam-sterilizer. 


tripod,  supply  the  heat.  In  the  cover  is  an  opening  for  a  ther- 
mometer. The  chest  is  usually  covered  with  felt.  When  the 
thermometer  registers  100°  C.  the  culture-medium  or  other  sub- 
stance to  be  sterilized  is  placed  in  the  steam  and  kept  there 
from  10  to  15  minutes,  or  longer,  as  required. 

Arnold's  steam  sterilizer  will  answer  every  purpose  of  the 
Koch  steam-chest.  It  is  cheaper,  also  requiring  less  fuel  to  keep 
it  going.  The  steam  does  not  escape,  but  is  condensed  in  the 
outer  chamber,     (rig.  16.) 


46 


ESSENTIALS  OF  BACTERIOLOGY 


The  autoclave  of  Chamberland  allows  a  temperature  of  120° 
C.  to  be  obtained,  and  is  much  used  in  Pasteur's  laboratory. 


Fig.  17. 


Chamberland's  Autoclave  with  pressure. 


Instead  of  sterilizing  for  a  long  time  at  once,  successive  steri- 
lization is  practised  with  nutrient  media,  so  that  the  albumen 
will  not  be  too  strongly  coagulated.  Fifteen  minutes  each  day 
for  three  days  in  succession. 


METHODS   OF   CULTURE, 


47 


Fractional  Sterilization  of  Tyndall.  Granted  that  so  many 
spores  originally  exist  in  the  object  to  be  sterilized,  it  is  sub- 
jected to  60°  0.  for  four  hours,  in  which  time  a  part  at  least  of 
those  spores  have  developed  into  bacteria,  and  the  bacteria 
destroyed  by  the  further  application  of  the  heat.  The  next  day 
more  bacteria  will  have  formed,  and  four  hours'  subjection  to 
60°  heat  will  destroy  them,  and  so  at  the  end  of  a  week,  using 
four  hours'  application  each  day,  all  the  spores  originally  present 
will  have  germinated  and  the  bacteria  destroyed. 


Fig.  18. 


Fig.  19. 


i1    1 

i' 

! 

i 

1  1 

i  i 

! 

LJ 

■I 

!i 

Wire-Cage. 


Cotton  plugged  Test-Tubes. 


Cotton  Plugs  or  Corks.  All  the  glass  vessels  (test-tubes,  flasks, 
etc.)  must  be  closed  with  cotton  plugs,  the  cotton  being  easily 
sterilized  and  preventing  the  entrance  of  germs. 

Tin-foil  may  be  used  to  cover  the  cotton,  or  caps  made  of 
india-rubber. 

Test-tubes.  New  test-tubes  are  washed  with  hydrochloric 
acid  and  water  to  neutralize  the  alkalinity  often  present  in 
fresh  glass.     They  are  then  well  washed  and  rubbed  with  a 


48         ESSENTIALS  OP  BACTERIOLOGY. 

brush,  placed  obliquely  to  drain,  and  when  dry  corked  with 
cotton  plugs.  Then  put  in  the  hot-air  oven  (little  wire-cages 
being  used  to  contain  them)  for  fifteen  minutes,  after  which  they 
are  ready  to  be  filled  with  the  nutrient  media.  (The  cotton 
should  fit  firmly  in  the  tube  and  extend  a  short  space  beyond  it.) 

Test-tubes  without  flaring  edges  are  more  desirable  since  the 
edges  can  easily  be  drawn  out  so  as  to  seal  the  tube. 

Instead  of  test-tubes,  ordinary  3  oz.  panel  medicine  bottles 
can  be  used  for  retaining  the  nutrient  media  and  cultures. 

According  to  late  investigations,  the  glass  tubes  become  suffi- 
ciently sterile  in  the  steam-chest  without  the  preliminary  sterili- 
zation in  the  dry  oven. 


CHAPTER  VIII. 

NUTRIENT   MEDIA. 


Of  the  many  different  media  recommended  and  used  since 
bacteriology  became  a  science,  we  can  only  describe  the  more 
important  ones  now  in  use.  Each  investigator  changes  them 
according  to  his  taste. 

Fluid  Media. 

Bouillon  (according  to  Loftier).  A  cooked  infusion  of  beef 
made  slightly  alkaline  with  soda  carbonate :  500  grammes  of 
finely-chopped  raw  lean  beef  are  placed  in  a  wide-mouthed  jar 
and  covered  with  1  litre  of  water ;  this  is  left  standing  twelve 
hours  with  occasional  shaking.  It  is  then  strained  through 
cheese  cloth,  the  white  meat  remaining  being  pressed  until  one 
litre  of  the  blood-red  meat-water  has  been  obtained.  The  meat- 
water  must  now  be  cooked,  but  before  doing  this,  in  order  to 
prevent  all  the  albumen  from  coagulating,  10  parts  of  peptone 
powder  and  5  parts  of  common  salt  are  added  to  every  1000 
parts  meat-water.  It  is  next  placed  in  the  steam-chest  or 
water-bath  for  three-quarters  of  an  hour. 

Neutralization.  The  majority  of  bacteria  grow  best  on  a 
neutral  or  slightly  alkaline  soil,  and  the  bouillon,  as  well  as 


NUTRIENT    MEDIA.  49 

other  media,  must  be  carefully  neutralized  with  a  sat.  sol.  of 
carbonate  of  soda.  Since  too  much  alkalinity  is  nearly  as  bad 
as  none  at  all,  the  soda  must  be  added  drop  by  drop  until  red 
litmus  paper  commences  to  turn  blue.  The  bouillon  is  then 
cooked  another  hour,  and  filtered  when  cold.  The  liquid  thus 
obtained  must  be  clearly  alkaline,  and  not  clouded  by  further 
cooking.  If  cloudiness  occur,  the  white  of  an  egg  and  further 
boiling  will  clear  the  same.  To  make  bouillon,  beef-extract  can 
be  used  instead  of  fresh  meat,  2  grammes  to  1  litre  of  water. 
This  is  boiled  with  5  grammes  of  salt  and  10  of  peptone,  neu- 
tralized as  above,  and  filtered  when  cold. 

Schultz's  Method  of  Neutralization. — A  more  accurate  method  of 
obtaining  the  required  reaction  is  to  use  an  alcoholic  solution 
(£  per  cent.)  of  phenolphthalein  as  an  indicator ;  a  few  drops  of 
this  are  mixed  with  10  c.c.  of  the  bouillon,  and  from  a  burette  a 
solution  of  caustic  soda  0.4  per  cent,  is  added  drop  by  drop 
until  a  faint  red  color  appears.  An  average  is  taken  from  three 
different  samples,  and  the  amount  of  soda  needed  for  the  entire 
quantity  of  bouillon  is  calculated  therefrom.  Glucose  broth, 
which  is  a  good  medium  for  anaerobic  organisms,  consists  of 
bouillon  to  which  1  to  2  per  cent,  of  grape-sugar  has  been 
added.  Glycerin  broth  is  bouillon  to  which  6  to  8  per  cent,  of 
glycerin  has  been  added  after  filtration. 

Sterilization  of  the  Bouillon.  Erlenmeyer  flasks  (little  conical 
glass  bottles)  or  test-tubes  plugged  and  properly  sterilized  are 
filled  one-third  full  with  the  bouillon,  and  placed  with  their 
contents  in  the  steam-chest.  They  are  left  in  steam  of  100°  C. 
one  hour  for  three  successive  days,  after  which  the  tubes  and 
bouillon  are  ready  for  use. 

Solid  Media.  The  knowledge  of  bacteria  and  germs  or  moulds 
settling  and  growing  upon  slices  of  potato  exposed  to  the  air,  led 
to  the  use  of  solid  media  for  the  artificial  culture  of  the  same. 
It  was  also  thus  learned  that  each  germ  tends  to  form  a  separate 
colony  and  remain  isolated. 

Potato-Cultures.  A  ripe  potato  with  a  smooth  skin  is  the 
best. 

Several  are  brushed  and  scrubbed  with  water  to  get  rid  of  the 
dirt  and  the  "eyes"  are  cut  out. 
4 


50 


ESSENTIALS    OF    BACTERIOLOGY. 


Next  placed  in  1  to  500  solution  of  bichloride  of  mercury  for 
£  hour.     Then  in  the  steam-chest  for  £  hour. 

In  the  meantime,  a  receptable  is  prepared  for  them.  This  is 
called  the  moist  chamber. 

Fig.  20. 


Moist  chamber  for  potatoes. 

The  moist  chamber  consists  of  two  large  shallow  dishes,  one, 
the  larger,  as  a  cover  to  the  other. 
These  dishes  are  washed  in  warm  distilled  water. 


Fig.  21. 


Method  of  slicing  potato.    (After  Woodhead  and  Hare.) 

A  layer  of  filter  paper  moistened  with  a  15  to  30  drops  of  1  to 
1000  bichloride  is  placed  in  the  bottom  of  the  glass  dish. 

The  operator  now  prepares  his  own  hands,  rolling  up  his  coat 
sleeves  and  carefully  washing  his  hands,  then  taking  a  potato 


NUTRIENT    MEDIA. 


51 


from  the  steam-oven  and  holding  it  between  his  thumb  and 
index  finger  in  the  short  axis,  he  divides  the  potato  in  its 
long  axis  with  a  knife  that  has  been  passed  through  the  flame. 
The  two  halves  are  kept  in  contact  until  they  are  lowered  into 
the  moist  chamber,  when  they  of  their  own  weight  fall  aside, 
the  cut  surface  uppermost.     They  are  then  ready  for  inoculation. 

Esmarch's  Cubes.  The  potato  is  first  well  cleaned  and  peeled. 
It  is  then  cut  in  cubes  £  inch  in  size. 

These  are  placed,  each  in  a  little  glass  dish  or  tray  and  then 
in  steam-chest  for  ^  hour,  after  which  they  are  ready  for  inocu- 
lation (the  dishes  first  having  been  sterilized  in  hot-air  oven). 

Test-tube  Potatoes.  Cones  are  cut  out  of  the  peeled  potato 
and  placed  in  test-tubes,  which  can  then  be  plugged  and  easily 
preserved. 

Roux's  test-tube  (Fig.  22),  specially  designed         Fig.  22. 
for  potato   cultures,  consists  of  a  tube  with  a 
small  constricted  portion  at  the  bottom,  in  which 
water  may  be  kept  to  keep  the  potato  moist. 

Manner  of  Inoculation.  With  a  platinum  rod 
or  a  spatula  (sterilized)  the  material  is  spread 
upon  one  of  the  slices,  keeping  free  of  the 
edges.  The  growth  on  this  first,  or  original, 
potato  will  be  quite  luxuriant,  and  the  individual 
colonies  often  difficult  to  recognize;  therefore 
dilutions  are  made.     (Fig.  23.) 

From  the  original  or  first  slice  a  small  portion, 
including  some  of  the  meat  of  the  potato,  is 
spread  upon  the  surface  of  a  second  slice, 
which  is  first  dilution.  From  this  likewise  a 
small  bit  is  taken  and  spread  on  a  third  slice,  or 
second  dilution,  and  here  usually  the  colonies 
will  be  sparsely  enough  settled  to  study  them  in  their  indi- 
viduality. 

This  is  the  principle  carried  on  in  all  the  cultivations.  It  is 
a  physical  analysis. 

Potato  and  Bread  Mash.  These  pastes  are  used  chiefly  in  the 
culture  of  moulds  and  yeasts.  Peeled  potatoes  are  mashed  with 
distilled  water  until  thick,  and  then  sterilized  in  flasks  f  of  an 
hour  for  three  successive  days. 


Tube  for  potato 
culture. 


52  ESSENTIALS    OP    BACTERIOLOGY. 

Fig.  23. 


Method  of  inoculation.    (Woodhead  and  Hare.) 

Bread  Mush.— Bread  devoid  of  crust,  dried  in  an  oven,  and 
then  pulverized  and  mixed  with  water  until  thick  and  sterilized 
as  above. 


CHAPTER  IX. 


SOLID   TRANSPARENT   MEDIA. 


Solid  Transparent  Media  are  materials  which  can  be  used  for 
microscopical  purposes  and  which  can  readily  be  converted 
into  liquids.     Such  are  the  gelatine  and  agar  culture  media. 

Gelatine.  Gelatine  is  obtained  from  bones  and  tendons,  and 
consists  chiefly  of  chondrin  and  gluten. 

The  French  golden  medal  brand  is  the  one  most  in  use,  found 
in  long  leaves  with  ribbed  lines  crossing  them. 

Koch-Loffler  10  per  cent.  Bouillon-Gelatine.    To  the  meat- 
water  as  made  for  the  bouillon  are  added 
100  grammes  gelatine, 
10        "         peptone, 
5        "        salt, 
to  each  1000  grammes  of  the  meat-water;  or  to  the  bouillon 
made  from  beef-extract  the  gelatine  is  added ;  this  is  placed  in 
a  flask  and  gently  heated  until  the  gelatine  is  dissolved. 

Neutralization  with  the  soda  and  then  cooking  in  water-bath 
or  carefully  boiled  over  flame  for  1  hour  or  more  until  the 


SOLID   TRANSPARENT   MEDIA, 


53 


liquid  seems  clear,  then  add  white  of  an  egg  and  boil  £  hour 
longer ;  the  egg  will  produce 


a  clearer  solution  and  save 
much  trouble.  A  small  por- 
tion, while  hot,  is  now  filtered 
into  a  test-tube  and  tested  for 
alkalinity,  and  then  re-heated 
several  times,  watching  if  a 
cloudy  ppt.  forms. 

If  the  fluid  remains  clear 
upon  cooling,  the  remainder  of 
the  material  can  be  filtered. 
It  must  be  accomplished 
while  hot,  else  the  gelatine 
will  coagulate  and  prevent 
further  filtration. 

This  can  be  carried  on 
either  by  keeping  hot  the  so- 
lution continually  in  water- 
bath,  and  only  filtering  a  small 
quantity  at  a  time  through 
the  filter,  or  keeping  the  filter 
itself  hot,  either  with  a  hot 
water  filter  or  placing  the 
filter  in  steam  chest.  (Fig. 
24.) 

Clouding  of  Gelatine.  If  the  gelatine  does  not  come  out  clear, 
or  becomes  turbid  on  cooling,  it  may  be  due  to  several  things— 

1.  The  filter-paper  too  thin  or  impure. 

2.  Too  strongly  alkaline. 

3.  Cooked  too  long  or  not  long  enough. 

The  addition  of  the  white  of  an  egg,  as  before  mentioned,  will 
often  clear  it  up  ;  if  this  avails  not,  re-filtering  several  times,  and 
attention  to  the  few  points  mentioned. 

Sterilizing  the  Gelatine.  The  gelatine  is  kept  in  little  flasks 
or  poured  at  once  into  sterile  test-tubes,  careful  not  to  wet  the 
neck  where  the  cotton  enters,  lest  when  cool  the  cotton  plug 
stick  to  the  tube. 

The  tubes  are  then  placed  in  steam-chest  for  three  successive 


Hot-water 


54         ESSENTIALS  OP  BACTERIOLOGY. 

days,  15  minutes  each  day  (or  in  water-bath  1  hour  a  day  for 
three  days).  Then  set  aside  in  a  temperature  of  15°  to  20°  C, 
and  if  no  germs  develop  and  the  gelatine  remains  clear,  it  can 
be  used  for  cultivation  purposes. 

Modifications.  The  amount  of  gelatine  added  to  the  meat- 
water  can  be  variously  altered,  and  instead  of  making  gelatine 
bouillon  milk,  blood,  serum,  urine,  and  agar  can  be  added. 
Glycerine  (4  to  6%)  is  a  common  addition,  and  sometimes 
reducing  agents  to  absorb  the  oxygen  are  mixed  with  it. 

Agar- Agar.  This  agent,  which  is  of  vegetable  origin,  derived 
from  sea-plants  gathered  on  the  coasts  of  India  and  Japan,  has 
many  of  the  properties  of  gelatine,  retaining  its  solidity  at  a 
much  higher  temperature;  it  becomes  liquid  at  90°  C.  and  con- 
geals again  at  45°  C.     Gelatine  will  liquefy  at  35°  C. 

It  is  not  affected  very  much  by  the  peptonizing  action  of 
the  bacteria — 38°  C.  is  the  temperature  at  which  most  patho- 
genic germs  grow  best. 

Preparation  of  Agar-Agar  Bouillon  or  Nutrient  Agar.  The 
ordinary  bouillon  is  first  made,  and  then  the  agar  cut  in  small 
pieces,  added  to  the  bouillon  (15  grammes  of  agar  to  1000 
grammes  bouillon.  It  is  allowed  to  stand  several  minutes  until 
the  agar  swells,  and  then  placed  in  water-bath  or  steam-chest 
for  six  hours  or  more.  It  is  then  neutralized,  very  little  of  the 
alkali  being  sufficient. 

A  white  of  an  egg  added,  and  boiled  for  several  hours  longer, 
when,  even  if  not  perfectly  clear,  it  is  filtered. 

The  filtering  process,  very  difficult  because  of  the  readiness 
with  which  the  agar  solidifies,  must  be  done  in  steam-chest  or 
with  hot-water  filter,  and  very  small  quantities  passed  through 
at  a  time,  changing  the  filter-paper  often. 

Cotton  can  be  used  instead  of  filter-paper,  or  filtering  entirely 
dispensed  with,  simply  decanting. 

A.s  agar  is  seldom  clear,  a  little  more  or  less  opaqueness  will 
not  harm.  The  test-tubes  are  filled  as  with  the  gelatine,  and 
sterilized  in  the  same  manner.  While  cooling,  some  of  the 
tubes  can  be  placed  in  a  slanting  position,  so  as  to  obtain  a  larger 
surface  to  work  upon. 

Water  of  condensation  will  usually  separate  and  settle  at  the 


SOLID   TRANSPARENT   MEDIA.  55 

bottom,  or  a  little  white  sediment  remain  encysted  in  the  centre ; 
this  cannot  easily  be  avoided,  nor  does  it  form  any  serious  obstacle. 

The  crude  agar  should  first  be  rinsed  in  water,  and  then  in 
5%  acetic  acid  and  clear  water  again,  to  rid  it  of  impurities. 
If  agar  is  boiled  thoroughly  over  a  hot  flame  or  in  an  auto- 
clave, it  can  be  filtered  much  more  readily.  The  main  point 
is  to  see  that  all  the  agar  is  dissolved. 

It  has  been  suggested  to  pour  the  hot  agar  into  high  cylin- 
drical glass  vessels  and  allow  it  to  cool  slowly  in  the  steam 
oven,  the  flame  having  been  gradually  lowered  and  then  turned 
out.  After  a  time  the  cloudy  portion  will  form  a  sediment  at 
the  bottom ;  the  agar  can  then  be  shaken  out  as  a  long  cylinder 
and  the  cloudy  portion  cut  off. 

The  Japanese  Method. — Yokote  prepares  agar  as  follows :  the 
meat  is  cooked  in  water  over  a  sand  bath  1?  hours.  Filtered, 
chopped  agar  is  then  added  and  the  mixture  cooked  1  hour 
longer ;  peptone  and  salt  added  next.  Neutralization.  After  the 
mixture  has  cooled  to  about  50°  C.  whites  of  2  eggs  are  added 
and  the  mixture  shaken  thoroughly. 

Again  the  mixture  is  placed  on  the  sand-bath  and  heated  to 
110°  C.  and  over  for  lj  to  2  hours,  and  then  filtered  through 
ordinary  filter-paper.  Yokote  claims  that  by  this  procedure 
the  agar  can  be  filtered  as  easily  as  bouillon  and  without  any 
loss.  (The  water  evaporated  in  boiling  must  be  added  before 
filtering.) 

Glycerine  Agar.  The  addition  of  4  to  6%  of  glycerine  to 
nutrient  agar  greatly  enhances  its  value  as  a  culture  medium. 

Gelatine-Agar.  A  mixture  of  5  per  cent,  gelatine  and  0.75 
per  cent,  agar  combines  in  it  some  of  the  virtues  of  both  agents. 

Blood  Serum.  Blood  serum  being  rich  in  albumen  coagulates 
very  easily  at  70°  C,  and  if  this  temperature  is  not  exceeded, 
a  transparent,  solid  substance  is  obtained  upon  which  the  ma- 
jority of  bacteria  develop,  and  some  with  preference. 

Preparation  of  Nutrient  Blood  Serum.  If  the  slaughter  of 
the  animal  can  be  supervised,  it  were  best  to  have  the  site  of 
the  wound  and  the  knife  sterilized,  and  sterile  flasks  to  receive 
the  blood  directly  as  it  flows. 

It  is  placed  on  ice  forty-eight  hours,  and  the  serum  is  drawn 


56 


ESSENTIALS  OF  BACTERIOLOGY. 


Fig.  25. 


out  with  sterile  pipettes  into  test-tubes;  these  are  placed  ob- 
liquely in  an  oven  where  the  temperature  can  be  controlled 
and  maintained  at  a  certain  degree.  See 
Fig.  26. 

Incubators  or  Brood-ovens.  Incubators 
or  brood-ovens  consist  essentially  of  a  double- 
walled  zinc  or  copper  chest,  the  space  between 
the  walls  being  filled  with  water. 

The  oven  is  covered  with  some  imperme- 
able material  to  prevent  the  action  of  the 
surrounding  atmosphere.  (Fig,  27.)  It  is 
supplied  with  a  thermometer  and  a  regu- 
lator. The  regulator  is  connected  with  a 
Bunsen  burner,  and  keeps  the  temperature 
at  a  certain  height. 

There  are  several  forms  of  regulators  in 
use,  and  new  ones  are  invented  continually. 
The  size  of  the  flame  in  some  is  regulated 
by  the  expansion  of  mercury,  which,  as  it 
rises,  lessens  the  opening  of  the  gas  supply. 
The  mercury  contracting  on  cooling  allows  more  gas  to  enter 
again.     (Fig.  28.) 

Koch  has  invented  a  safety  burner,  by  which  the  gas  supply  is 
shut  oft*  should  the  flame  accidentally  have  gone  out. 

Coagulation  of  Blood  Serum.  The  tubes  of  blood  serum 
having  been  placed  in  the  oven,  are  kept  at  a  tempera- 
ture of  65°  to  68°  C,  until  coagulation  occurs ;  then  removed 
and  sterilized. 

Sterilization  of  Blood  Serum.  The  tubes  are  placed  3  to  4 
days  in  incubation  at  58°  C,  and  those  tubes  which  show  any 
evidences  of  organic  growth  are  discarded. 

If  now,  at  the  end  of  a  week,  the  serum  remains  sterile  at  the 
ordinary  temperature  of  the  room,  it  can  be  used  for  experi- 
mental purposes. 

Perfectly  prepared  blood  serum  is  transparent,  of  a  gelatine- 
like consistence,  and  straw-color.  It  will  not  liquefy  by  heat, 
though  bacteria  can  digest  it.  Water  of  condensation  always 
forms,  which  prevents  the  drying  of  the  serum.     Blood  serum 


Flask  to  receive  blood 
serum. 


SOLID   TRANSPARENT    MEDIA. 


57 


may  be  prepared  in  a  shorter  way  by  coagulating  the  serum  at 
a  temperature  short  of  boiling-point.  Sterilization  is  completed 
in  three  days  by  exposing  the  tubes  to  a  temperature  of  about 
90°  C.  each  day  for  five  minutes.  Tubes  so  prepared  are  opaque 
and  white. 


Fro.  26. 


Thermostat  for  blood  serum. 


Preservation  of  Blood  Serum  in  Liquid  State.  Kirchner  advises 
the  use  of  chloroform.  To  a  quantity  of  serum  in  a  well-stop- 
pered flask  a  small  amount  of  chloroform  is  added — enough  to 
form  about  a  2  mm.  layer  on  the  bottom.  If  the  chloroform  is 
not  allowed  to  evaporate,  the  serum  remains  sterile  for  a  long 
time.  When  needed  for  use  test-tubes  are  filled  and  placed  in 
a  water-bath  at  50°  C.  until  all  chloroform  has  been  driven  off 
(determined  by  absence  of  characteristic  odor);  the  serum  is 
then  solidified  and  sterilized  as  in  the  ordinary  way. 


58 


ESSENTIALS  OP  BACTERIOLOGY. 


Human  blood  serum  derived  from  placenta,  serum  from  as- 
citic fluid  and  ovarian  cysts,  is  prepared  in  a  similar  manner 
to  the  above. 

Blood  Coagulum,  suggested  by  the  author,  is  the  blood  itself 
(not  the  serum  only)  coagulated  in  test-tubes.  It  is  dark  brown 
in  color  and  allows  some  colonies  of  bacteria  to  be  more  visi- 
ble. It  requires  less  time  to  prepare,  and  is  not  so  likely  to 
become  contaminated  as  when  the  serum  is  used. 


Fig.  27. 


Fig.  28. 


Babe's  incubator.  Thermo-regulators. 

Loffler's  Blood  Serum  Mixture  (see  p.  111). 

Peptone  Solution.  (Dunham's.)  Sodium  chlorid,  0.5;  pep- 
tone, 1 ;  water,  100.  Boil,  filter,  and  sterilize.  Useful  to  detect 
presence  of  indol. 

Other  Nutrient  Media.  Milk,  urine,  decoctions  of  various 
fruits  and  plants,  and  lately  for  cultivating  anaerobic  bacteria, 
eggs. 


SOLID   TRANSPARENT    MEDIA.  59 

Many  combinations  of  the  preceding  are  also  in  use,  such  as 
glucose-agar,  glucose  gelatin,  blood-  or  serum-agar;  and  litmus 
is  often  added  to  media  to  show  changes  in  reaction  during 
bacterial  growth. 

Dunham's  Rosalie  Acid  Solution. 

Peptone  sol.  (Dunham)      ....     100  c.c. 
2  per  cent.  sol.  rosalic  acid         .        .        .0.5  gr. 
Alcohol  (80  per  cent.)         ....     100  c.c. 
M.    To  detect  acids  and  alkalies. 

Eisner's  Medium  (for  typhoid).  (Iodo- potass. — Potato-gelatin.) 
Five  hundred  grammes  of  (peeled  and  washed)  potatoes  are 
mashed  and  pressed  through  a  fine  cloth.  The  juice  is  allowed 
to  settle,  is  filtered,  and  after  1  hour's  cooking  has  added  to  it  10 
per  cent,  gelatin;  then  2\  c.c.  ^  normal  sodic  hydrate  solu- 
tion, and  finally  1  per  cent,  potassic  iodid. 

Typhoid  Medium  of  Hiss.  This  consists  of  a  slightly  acid 
mixture  of  gelatin  and  agar,  beef-extract,  sodium  chloride,  and 
dextrose,  used  in  different  proportions  for  plate  and  tube  cul- 
tures. It  is  semi-solid  in  character  and  facilitates  the  identi- 
fication of  the  motile  typhoid  bacilli  which  produce  a  uniform 
clouding  through  the  medium  in  tubes. 

Urine  Media  (Gonococci). 

Urine  (sterile  taken)     .        .        .        ,        .1  part. 
2  per  cent,  agar  solution     .        .        .        .     1     " 

Fresh  Egg  Cultures,  after  Huppe.  The  eggs  in  the  shell  are 
carefully  cleaned,  washed  with  sublimate,  and  dried  with  cotton. 

The  inoculation  occurs  through  a  very  fine  opening  made  in 
the  shell  with  a  hot  platinum  needle ;  after  inoculation,  the  open- 
ing is  covered  with  a  piece  of  sterilized  paper,  and  collodion. 

Boiled  Eggs.  Eggs  boiled,  shell  removed  over  small  portion, 
and  the  coagulated  albumen  stroked  with  the  material. 

Guinea-pig  Bouillon.  The  flesh  of  guinea-pigs  as  well  as  that 
of  other  experiment-animals  is  used  instead  of  beef  in  the  prep- 
aration of  bouillon,  for  the  growth  of  special  germs. 

The  extracts  of  different  organs  have  been  added  to  the 
various  media  for  experimentation. 


60 


ESSENTIALS  OF  BACTERIOLOGY, 


CHAPTER  X. 

INOCULATION  OF  GELATINE  AND  AGAR. 

Glass  Slide  Cultures.  Formerly  the  gelatine  was  poured  on 
little  glass  slides  such  as  are  used  for  microscopical  purposes, 
and  after  it  had  become  hard,  inoculated  in  separate  spots  as 
with  potatoes. 


Fig.  29. 


Manner  of  holding  tubes  for  inoculation:  a, tube  with  material;  b,  tube  to  be 
inoculated  ;  c,  cotton  plugs.    (After  Woodhead  and  Hare.) 

Test  Tube  Cultures.    The  gelatine,  agar,  or  blood  serum  having 
solidified  in  an  oblique  position,  is  smeared  on  the  surface  with 


INOCULATION     OF    GELATINE    AND    AGAR.  61 

the  material  and  the  growth  occurs,  or  the  medium  is  punctured 
with  a  stab  of  the  platinum  rod  containing  the  material.  The 
first  is  called  a  stroke  or  smear  culture,  the  second  a  stab  or  thrust 
culture.  In  removing  the  cotton  plugs  from  the  sterile  tubes  to 
carry  out  the  inoculation,  the  plugs  should  remain  between  the 
lingers  in  such  a  way  that  the  part  which  comes  in  contact  with 
the  mouth  of  the  tube  will  not  touch  anything. 

After  the  needle  has  been  withdrawn  the  plugs  are  re-inserted 
and  the  tubes  labelled  with  the  kind  and  date  of  culture. 

Plate  Cultures.  Several  tubes  of  the  culture  medium  are 
made  liquid  by  heating  in  water  bath,  and  then  inoculated  with 
the  material  as  follows  .  A  looped  platinum  needle  is  dipped 
into  the  material  and  then  shaken  in  the  tube  of  liquid  media, 
(gelatine,  agar,  etc.). 

This  first  tube  is  called  original.  From  this  three  drops  (taken 
with  the  looped  platinum  rod)  are  placed  in  a  second  tube,  the 
rod  being  shaken  somewhat  in  the  gelatine  or  agar ;  this  is 
labeled  first  dilution  (a  colored  pencil  is  useful  for  such  markings). 
From  the  first  dilution  three  drops  are  taken  into  a  third  tube, 
which  becomes  the  second  dilution.    (Fig.  29.) 

The  plugs  of  cotton  must  be  replaced  after  each  inoculation, 
and  during  the  same  must  be  carefully  protected  from  contami- 
nation. 

To  hasten  the  procedure  and  lessen  the  danger  of  contamina- 
tion, the  tubes  can  be  held  in  one  hand  aside  of  each  other,  each 


Fig.  30. 


Manner  of  holding  plugs. 

plug  opposite  its  tube.     They  are  now  ready  for  spreading  on 
glass  plates. 


62 


ESSENTIALS  OF  BACTERIOLOGY. 


Glass  Plates. 


Fig.  31. 


z^Ek? 


The  larger  the  surface  over  which  the  nutrient 
medium  is  spread  the  more  isolated  will 
the  colonies  be  ;  window  glass  cut  in  rec- 
tangular plates  Gx4  inches  in  size  is  used  ; 
about  ten  such  plates  are  cleaned  with  dry 
towel  and  placed  in  a  small  iron  box  or 
wrapped  in  paper ;  and  sterilized  in  the 
hot-air  oven  at  a  temperature  of  150°  C. 
for  ten  minutes.     (Fig.   31.)    When  the 
plates  have  cooled  they  are  placed  upon 
an  apparatus  designed  to  cool  and  so- 
lidify the   liquid    media,   which  is   now 
poured  upon  the  plates  from  the  inocu- 
lated test-tubes. 
Nivellier  Leveling  and  Cooling  Apparatus.    Ice  and  water 
are  placed  in  a  shallow  round  glass  tray  ;  on  top  of  this  a  square 
plate  of  glass,  upon  which  the  culture  plate  is  placed,  and  cov- 
ering this  a  bell-glass. 

The  whole  is  upon  a  low,  wooden  tripod,  the  feet  of  which 
can  be  raised  or  lowered,  and  a  little  spirit-level  used  to  adjust 
it.  (Fig.  32.)  The  glass  plate  taken  out  of  the  iron  box  is  placed 
under  the  bell-glass.  The  tube  containing  the  gelatine  is  held 
in  the  flame  a  second  to  singe  the  cotton  plug  to  free  it  from  dust, 
and  the  plug  removed,  the  edges  of  the  tube  again  flamed,  the  bell- 
glass  lifted,  and  the  inoculated  gelatine  carefully  poured  on  the 
plate,  leaving  about  one-third  inch  margin  from  the  borders  ;  the 


Iron  box  for  glass  plates. 


Fig.  32. 


Nivellier  leveling  and  cooling  apparatus. 

lips  of  the  tube  being  sterile  can  be  used  to  spread  the  media 


INOCULATION  OF  GELATINE  AND  AGAR. 


63 


evenly.  If  the  plate  is  at  all  cool,  the  fluid  will  solidify  as  it  is 
being  spread.  The  glass  cover  is  replaced  until  the  gelatine  or 
agar  is  quite  solid  to  prevent  contamination. 

Fig.  33. 


Moist  chamber  with  plates  on  benches. 

When  the  gelatine  is  congealed,  the  plate  is  placed  upon  a 
little  glass  bench  or  stand  in  the  moist  chamber. 

The  Moist  Chamber  Prepared  Out  of  Two  Glass  Dishes,  as  for 
the  Potato- Cultures.  •  The  glass  benches  are  so  arranged  that 
one  stands  upon  the  other.  In  order  to  avoid  confusion,  a  slip 
of  paper  with  a  number  written  on  it  is  placed  on  the  bench  be- 
neath each  plate.  As  the  original  or  first  plate  would  have  the 
colonies  developed  in  greatest  profusion,  it  is  placed  the  first 
day  on  the  topmost  bench  ;  but,  since  the  colonies  would  be 
likely  to  overrun  the  plate  and  allow  the  gelatine  to  drop  on  the 
lower  plates,  it  is  best,  as  soon  as  evidences  of  growth  appear, 
to  place  it  below,  and  watch  the  third  plate  or  second  dilution 
for  the  characteristic  colonies,  forgetting  not  all  this  time  to 
change  the  numbers  accordingly. 

The  date  of  culture  and  the  name  can  be  written  upon  the 
moist  chamber. 

Petri  Saucers.  Agar  hardens  very  quickly,  even  without  any 
especial  means  for  cooling,  and  it  does 
not  adhere  very  well  to  the  glass.  There- 
fore it  is  better  to  follow  the  method  of 
Petri  and  use  little  shallow  glass  dishes, 
one  covering  the  other.  They  are  first 
sterilized  by  dry  heat,  and  then  the  in- 
oculated gelatine  or  agar  is  poured  into 
the  lower  dish,  covered  by  the  larger  one, 
and  placed  in  some  cool  place,  different  saucers  being  used  for 
each  dilution. 


Fig.  34. 


Petri  saucers. 


64  ESSENTIALS   OF    BACTERIOLOGY. 

This  method  is  very  useful  for  transportation ;  the  saucers 
can  be  viewed  under  microscope  similar  to  the  glass  plates,  and 
have  almost  entirely  superseded  them. 

Esmarch's  Tubes,  or  Rolled  Cultures.  This  method,  especially 
used  in  the  culture  of  anaerobic  germs,  consists  in  spreading  the 
inoculated  gelatine  upon  the  inner  walls  of  the  test  tube  in 
which  it  is  contained  and  allowing  it  to  congeal.  The  colonies 
then  develop  upon  the  sides  of  the  tube  without  the  aid  of 
other  apparatus.  The  method  is  useful  whenever  a  very  quick 
and  easy  way  is  required.  The  rolling  of  the  tube  is  done  under 
ice-water  or  running  water  from  the  faucet.  The  tube  is  held 
a  little  slanting,  so  as  to  avoid  getting  too  much  gelatine  around 
the  cotton  plug. 

The  tubes  can  be  placed  directly  under  the  microscope  for 
further  examination  of  the  colonies. 

It  is  almost  impossible  to  separate  certain  organisms,  such  as 
the  tubercle  bacillus  and  pneumococcus,  from  mixed  cultures 
by  ordinary  plate  methods,  and  the  plan  of  producing  the  dis- 
ease in  animals  by  inoculation,  and  then  obtaining  the  organ- 
ism in  pure  culture,  has  to  be  employed. 

Spored  organisms  may  also  be  separated  from  others  by  boil- 
ing the  mixture  for  a  few  minutes,  when  all  the  non-spored 
forms  will  perish,  and  only  the  spores  remain  to  germinate 
subsequently. 


CHAPTER  XL 

THE  GROWTH  AND  APPEARANCES  OF  COLONIES. 

Macroscopic.  Depending  greatly  upon  the  temperature  of 
the  room,  which  should  be  about  65°  C,  the  colonies  develop 
so  as  to  be  visible  to  the  naked  eye  in  two  to  four  days.  Some 
require  ten  to  fourteen  days,  and  others  grow  rapidly,  covering 
the  third  dilution  in  thirty-six  hours.  The  plate  should  be 
looked  at  each  day. 

The  colonies  present  various  appearances,  from  that  of  a 
small  dot,  like  a  fly-speck,  to  that  resembling  a  small  leaf. 


GROWTH    AND    APPEARANCE   OF   COLONIES. 


65 


Some  are  elevated,  some  depressed,  and  some,  like  cholera,  cup- 
shaped — umbilicated. 


Fig.  35. 


Staphylococcus  pyogenes  aureus :  colony  two  days  old,  seen  upon  an  agar-agar  plate; 
X  40  (Heim). 


Then  they  are  variously  pigmented.  Some  liquefy  the  gela- 
tine speedily,  others  not  at  all.  The  appearances  of  a  few  are 
so  characteristic  as  to  be  recognized  at  a  glance. 

Microscopic.  We  use  a  low-power  lens,  with  the  abbe  nearly 
shut  out,  that  is  the  narrowest  blender.  The  stage  of  the 
microscope  should  be  of  such  size  as  to  carry  a  culture  plate 
easily  upon  it. 

The  second  dilution  or  third  plate  is  usually  made  use  of,  that 
one  containing  the  colonies  sufficiently  isolated. 

These  isolated  ones  should  be  sought  for,  and  their  appearances 
well  noticed. 

There  may  be  two  or  three  forms  from  the  same  germ,  the 
difference  due  to  the  greater  or  less  amount  of  oxygen  that  they 
have  received,  or  the  greater  or  less  amount  of  space  that  they 
have  had  to  develop  in. 

The  microscopic  picture  varies  greatly  ;  now  it  is  like  the 
gnarled  roots  of  a  tree,  and  now  like  bits  of  frosted  glass  ;  the 
pictures  are  very  characteristic,  and  the  majority  of  bacteria 
can  be  told  thereby.     (Fig.  32.) 


66 


ESSENTIALS  OP  BACTERIOLOGY. 


Impression  or  "Klatsch"  Preparations.  In  order  to  more 
thoroughly  study  a  certain  colony  and  to  make  a  permanent 
specimen  of  the  same,  we  press  a  clean  cover-glass  upon  the 
particular  colony,  and  it  adheres  to  the  glass.     It  can  then  be 


Microscopic  appearances 
of  colonies. 


Fig.  37. 


Klatsch  preparations. 


stained  or  examined  so.  The  Germans  give  the  name  oi 
"Klatsch"  to  such  preparations.  Many  beautiful  pictures  can 
be  so  obtained. 

Fishing.  To  obtain  and  examine  the  individual  members  oi 
a  particular  colony  the  process  of  fishing,  as  it  is  called,  is 
resorted  to. 

The  colony  having  been  placed  under  the  field  of  the  micro- 
scope, a  long  platinum  needle,  the  point  slightly  bent,  is  passed 
between  the  lens  and  the  plate  so  as  to  be  visible  through  the 
microscope,  then  turned  downward  until  the  colony  is  seen  to 
be  disturbed,  and  the  needle  is  dipped  into  the  colony.  This 
procedure  must  be  carefully  done,  lest  a  different  colony  be 
disturbed  than  the  one  looked  at,  and  an  unknown  or  unwanted 
germ  obtained. 

After  the  needle  has  entered  the  particular  colony,  it  is  with- 
drawn, and  the  material  thus  obtained  is  further  examined  by 
staining  and  animal  experimentation.  The  bacteria  are  then 
again  cultivated  by  inoculating  fresh  gelatine,  making  stab  and 
stroke  cultures. 


CULTIVATION    OF    ANAEROBIC     BACTERIA 


67 


It  is  necessary  to  transfer  the  bacteria  to  fresh  gelatine  about 
every  six  weeks,  lest  the  products  of  growth  and  decay  given 
off  by  the  organisms  destroy  them. 


CHAPTER  XII. 


CULTIVATION    OF    ANAEROBIC   BACTERIA. 


Special  methods  are  necessary  for  the  culture  of  the  anaerobic 
variety  of  bacteria  in  order  to  procure  a  space  devoid  of  oxygen. 

Liborius's  High  Cultures.  The  tube  is  filled  about 
|  full  with  gelatine,  which  is  then  steamed  in  a  water 
bath  and  allowed  to  cool  to  40°  C,  when  it  is  inocu- 
lated by  means  of  a  long  platinum  rod  with  small 
loop,  the  movement  being  a  rotary  vertical  one,  and 
the  rod  going  to  the  bottom  of  the  tube. 

The  gelatine  is  next  quickly  solidified  under  ice ; 
very  little  air  is  present.  The  anaerobic  germs  will 
grow  from  the  bottom  upward,  and  any  aerobins 
present  will  develop  first  on  top,  this  method  being 
one  of  isolation. 

From  the  anaerobic  germs  grown  in  the  lower  part, 
a  stab  culture  is  made  into  another  tube  containing 
f  gelatine,  the  material  being  obtained  by  breaking 
test-tube  with  the  culture. 

Hesse's  Method.  A  stab  culture  having  been  made 
with  anaerobic  germs,  gelatine  in  a  semi-solid  condi- 
tion is  poured  into  the  tube  until  it  is  full,  thus  dis- 
placing the  air.     (Fig.  39.) 

Esmarch's  Method.  Having  inoculated  a  tube  with     Libo™s's 
the  microbe,  the  gelatine  is  rolled  out  on  the  walls  of 
the  tube,  a  "  roll  culture,"  and  the  rest  of  the  interior  filled  with 
gelatine,  the  tube  being  held  in  ice  water.    The  colonies  develop 
upon  the  sides  of  the  tube  and  can  be  examined  microscopically. 

Gases  like  Hydrogen  to  replace  the  Oxygen.  Several  ar- 
rangements for  passing  a  stream  of  hydrogen  through  the 
culture: — 


68 


ESSENTIALS    OP     BACTERIOLOGY. 


Frankel  puts  in  the  test  tube,  a  rubber  cork  containing  two 
glass  tubes,  one  reaching  to  the  bottom  and  connected  with  a 
hydrogen  apparatus,  the  other  very  short,  both  bent  at  right 
angles.  When  the  hydrogen  has  passed  through  ten  to  thirty 
minutes,  the  short  tube  is  annealed  and  then  the  one  in  connec- 
tion with  the  hydrogen  bottle,  and  the  gelatine  rolled  out  upon 
the  walls  of  the  tube.  (Fig.  40.)  Hiippe  uses  eggs  as  described 
in  Chapter  IX. 

Fig.  39.  Fig.  40.  Fig.  41. 


Hesse's  method. 


Frankel's  method. 


Buchner's  method. 


Use  of  iErobic  Bacteria  to  remove  the  Oxygen.  Roux  inocu- 
lates an  agar  tube  through  a  needle  thrust  after  which  semi- 
solid gelatine  is  poured  in  on  top.  When  the  gelatine  has  solidi- 
fied, the  surface  is  inoculated  with  a  small  quantity  of  bacillus 
subtilis  or  some  other  aerobic  germ.  The  subtilis  does  not  allow 
the  oxygen  to  pass  by,  appropriating  it  to  itself. 


CULTIVATION   OF   ANAEROBIC    BACTERIA 


69 


Buchner's  Method.     The  test  tube  containing  the  culture  is 
placed  within  a  larger  tube,  the  lower  part  of  which  contains 
an  alkaline  solution  of  pyrogallic  acid. 
The  tube  is  then  closed  with  a  rubber 
stopper.     (Fig.  41.) 

Botkin's  Method.  Petri  dishes,  un- 
covered, are  placed  on  a  rack  under  a 
large  bell-jar,  into  which  hydrogen  gas 
is  conducted.  Alkaline  pyrogallic  acid 
is  placed  in  the  upper  and  lower  dishes 
to  absorb  what  oxygen  remains. 

Wright's  Method.  Applicable  to 
both  fluid  and  solid  media.  After  in- 
oculating the  test-tube,  the  plug,  which 
must  be  of  absorbent  cotton,  is  cut  off 
flush  with  the  extremity  of  the  tube 
and  pushed  inward  for  a  distance  of  1 
cm.  It  is  then  impregnated  with  1  c.c. 
of  a  watery  solution  of  pyrogallic  acid 
and  1  c.c.  of  5  per  cent,  sodium  hydrate 
solution.  A  tightly  fitting  rubber  stop- 
per is  inserted,  and  the  tube  is  then 
ready  for  incubation. 

Park's  Method.  An  Erlenmeyer 
flask  containing  the  medium  to  be  used 
is  boiled  in  a  water-bath  from  ten  to 
fifteen  minutes  to  drive  off  dissolved 
oxygen,  quickly  cooled,  and  inoculated. 
Hot  melted  paraffme  is  then  poured 
into  the  flask,  which  forms  a  layer  over 
the  medium  and  on  congealing  pro- 
vides an  air-tight  seal  which  does  not 
adhere  to  the  glass  so  closely  as  to  pre- 
vent the  escape  of  any  gases  formed  by  the  bacterial  growth. 


Fin.  42.— Wright's  method 
for  the  cultivation  of  anae- 
robes. 


70  ESSENTIALS   OF   BACTERIOLOGY. 


CHAPTER  XIII. 

INFECTION. 

How  Bacteria  Cause  Disease.  Many  theories  have  been 
put  forward  to  explain  the  action  of  bacteria  in  causing  dis- 
ease, but  only  a  few  of  the  more  important  ones  can  be  taken 
up  here. 

What  are  the  Conditions  Necessary  to  Produce  Infection? 

First.  As  to  the  Infective  Agent.  The  organism  must  have  the 
power  to  produce  disease.  It  must,  in  other  words,  be  pathogenic.  A 
non-pathogenic  bacterium  under  certain  conditions  may  cause 
disease,  but  this  is  not  an  infectious  disease ;  it  is  rather  a  tox- 
emia, and  is  due  to  the  absorption  of  poisons  generated  outside 
of  the  body.  It  must  be  parasitic — have  the  power  of  growing 
within  the  body  of  an  animal. 

Essentially  an  infectious  disease  is  a  toxemia,  because  it 
depends  upon  poisons  or  toxins  produced  in  the  body.  Para- 
sitic or  infectious  bacteria  cause  disease  by  growing  in  the  animal 
organism  and  generating  products  therein  which  are  toxic. 
Saprophytic  bacteria  grow  outside  of  the  animal  organism  in  dead 
matter,  decaying  particles,  etc.,  and  they  may  give  rise  to  prod- 
ucts which  also  are  toxic  to  the  animal  economy. 

Second.  The  toxins  or  poisons  elaborated  must  be  present  in 
sufficient  amount.  Undoubtedly  each  animal  organism  is  a  law 
unto  itself  in  regard  to  the  amount  of  poison  it  will  tolerate 
before  disease  is  actually  produced.  The  period  of  incubation 
can  be  explained  on  the  supposition  that  the  germ  requires  so 
much  time  to  elaborate  the  amount  of  toxin  necessary.  This 
time  period  varies  with  different  organisms,  some  carrying  the 
toxin  with  them  at  the  time  of  entry. 

Third.  The  animal  infected  must  be  susceptible.  Susceptibility 
varies  in  different  species  of  animals,  in  different  members  of 
the  same  species,  in  the  same  individual  at  different  times,  and 
in  the  same  individual  to  the  different  forms  of  disease  germs. 
Susceptibility  may  be  natural  to  the  race,  it  may  be  acquired,  it 
may  be  inherited.  Mice  are  naturally  susceptible  to  anthrax. 
Acquired  susceptibility  occurs  upon    exposure   to   conditions 


INFECTION.  71 

which  lower  vitality,  as  hunger,  cold,  advanced  age,  and  sur- 
gical shock.  Inherited  susceptibility  is  a  less  important  factor 
now  than  formerly.  Many  diseases  were  at  one  time  considered 
inherited  which  now  are  known  to  be  acquired  during  the  life- 
time, of  an  individual.  Still,  certain  physical  characteristics, 
such  as  narrow  chest,  mouth-breathing,  etc. — clearly  inheritable 
characters — predispose  to  disease.  Given  a  susceptible  individ- 
ual and  an  infective  microorganism  producing  toxins  in  suffi- 
cient amount,  disease  is  certain  to  result. 

Local  Effects  of  Bacteria.  By  mechanical  obstruction  from 
rapid  growth,  thrombosis,  with  its  consequences,  may  occur. 
Destruction  of  a  part  of  the  cells  of  a  tissue  with  necrosis  can 
arise  from  irritation,  as  from  a  foreign  body. 

General  Effects.  Sapremia,  when  toxic  products  of  local 
suppuration  are  absorbed  into  the  system.  Septicemia,  when  the 
infective  agent  itself  enters  the  blood-stream  and  causes  general 
disturbance. 

Suppurative  bacteria  are  those  which  give  rise  to  inflamma- 
tion and  suppuration  locally  at  the  point  of  entrance,  and 
secondarily  through  metastasis.  Any  organism  may  cause 
suppuration,  but  a  certain  number  are  peculiarly  inclined  to 
give  rise  to  pus,  and  are  known  as  pyogenic  organisms. 

Infective  bacteria  are  as  a  rule  specific,  the  particular  toxin 
having  a  specific  action  and  causing  a  disease  peculiar  to  the 
microorganism.  Thus  typhoid  fever  is  a  disease  distinctly 
different  from  tuberculosis ;  the  infective  organisms  are  distinct 
and  the  poisons  they  produce  have  specific  characteristics. 

The  Nature  of  Toxins.  Very  similar  to  the  venom  of  ser- 
pents; highly  poisonous  in  minute  doses  (y^  gramme  of 
tetanus  toxin  will  kill  a  horse  weighing  600  kilos  (1200  pounds,). 
At  first  toxins  were  called  ptomaines,  or  cadaveric  alkaloids ;  but 
this  term  is  applied  now  to  such  poisons  as  have  a  basic  nature 
and  arise  in  decomposing  meat,  cheese,  and  cream  as  a  result 
of  chemical  change  in  the  material,  the  bacteria  causing  the 
change.  Then  they  were  called  toxalbumins,  and  were  supposed 
to  belong  to  an  albumin  series;  but  when  the  bacteria  are  grown 
in  non-albuminous  media  the  toxins  correspond  more  in  their 
chemical  composition  to  a  ferment,  and  therefore  it  is  supposed 


72  ESSENTIALS    OF    BACTERIOLOGY. 

that  the  albumin  part  of  the  toxin  is  furnished  by  the  blood  or 
albuminous  media  in  which  it  is  formed.  The  term  toxin  is  to 
be  preferred  in  speaking  of  bacterial  poisons. 

Toxins  may  be  of  two  sorts :  (a)  Chiefly  within  the  bodies  of 
the  bacteria,  so  that  they  are  set  free  by  the  disintegration  of 
the  organisms.  This  group  comprises  most  of  the  pathogenic 
bacteria  and  must  be  combatted  by  the  use  of  antibacterial  sera. 
(b)  The  poisons  seem  to  be  excreted  by  the  bacteria  and  are 
found  in  the  surrounding  medium.  Antitoxic  sera  are  applica- 
ble to  this  group,  which  includes  the  bacilli  of  diphtheria  and 
tetanus.  Welch  has  suggested  that  even  bacteria  which  do  not 
appear  to  form  toxins  in  artificial  cultures  may  do  so  in  the 
human  body.  In  the  effort  to  adapt  themselves  to  their 
environment  and  resist  the  hostile  agencies  of  the  body  they 
produce  the  poisons  we  call  toxins.  (For  method  of  produc- 
tion of  an  antitoxin,  see  article  on  Diphtheria.) 


CHAPTER  XIV. 

IMMUNITY. 


Immunity,  as  distinguished  from  susceptibility,  is  merely  a  rela- 
tive term,  as  no  animal  is  absolutely  immune  under  all  condi- 
tions. It  is  merely  less  susceptible,  and  some  animals  are  by 
nature  or  can  by  artificial  means  be  rendered  so  slightly  sus- 
ceptible that  to  all  practical  purposes  they  are  immune— that  is, 
capable  of  resisting  an  attack  of  the  particular  disease  against 
which  they  are  said  to  be  immune. 

Natural  Immunity.  The  goat  and  dog  are  considered 
naturally  immune  to  tuberculosis.  Algerian  sheep  are  resistant 
to  anthrax,  other  varieties  are  susceptible. 

The  field  mouse  is  susceptible  to  glanders,  the  white  mouse 
is  ordinarily  immune.  House  mice  are  susceptible  to  mouse 
septicemia,  field  mice  are  immune. 

Acquired  Immunity.  Immunity  can  be  acquired  in  many 
ways.     Active  and  passive  immunity  are  varieties. 

Active  immunity  can  be  acquired  from  an  attack  of  the  disease; 


IMMUNITY.  73 

such  infectious  diseases  as  measles,  scarlatina,  and  whooping- 
cough  usually  confer  immunity  from  future  attacks.  Some 
diseases  render  the  individual  immune  for  only  a  short  period. 

Immunity  from  Inoculation  with  Attenuated  or  Weak- 
ened Cultures  of  Bacteria.  Vaccination  is  an  example.  Haff- 
kine's  cholera  vaccines  and  Pasteur's  vaccines  of  anthrax  and 
chicken  cholera  are  likewise  examples  of  this  method. 

Attenuation  is  produced  as  follows  :  Successive  cultivation  in 
artificial  media  destroys  the  virulence  of  bacteria.  Old  cultures 
are  less  virulent  than  fresh  ones.  Virulence  is  lessened  by 
passing  the  cultures  through  animals  that  are  less  susceptible 
or  entirely  immune.  The  cautious  use  of  chemicals  and  sun- 
light lessens  virulence.  Heat  is  an  effective  agent.  An  anthrax- 
culture  exposed  to  a  temperature  of  42.6°  C.  for  twenty  days 
will  prove  destructive  only  to  animals  no  larger  than  mice. 
Prolonged  exposure  to  oxygen  weakens  the  germs. 

Immunity  Through  Inoculations  of  Small  Doses  of  very 
Virulent  Microorganisms.  A  graduated  resistance  to  the 
disease  is  reached  somewhat  after  nature's  method.  By  succes- 
sive inoculations  with  increased  doses  of  the  virus  an  immunity 
is  often  reached  sufficient  to  withstand  ten  times  the  lethal 
dose.     A  poison-habit  is  thus  acquired. 

Increased  Virulence  is  produced  as  follows :  The  cultures 
may  be  greatly  increased  in  virulence  by  successive  cultivation 
through  animals,  and  gradually  changing  from  smaller  animals 
to  larger,  until  an  amount  of  the  culture  that  at  the  outset 
would  not  destroy  a  guinea-pig  becomes  finally  virulent  for 
chickens  and  dogs. 

Immunity  Through  Injections  of  the  Sterilized  Products  of 
Bacteria.  Cultures  sterilized  by  heat  or  nitration  through 
germ-filters  still  contain  the  chemical  products  of  bacteria,  the 
toxins ;  and  when  these  are  injected  in  gradually  increased  doses 
the  same  immunity  is  obtained  as  with  the  bacteria  themselves. 

Passive  Immunity.  The  blood-serum  and  tissues  generally 
of  animals  rendered  immune  in  the" ways  described  above,  when 
injected  into  susceptible  animals  render  them  immune  against 
the  same  infection.  This  has  been  called  passive  immunity,  but 
there  is  no  strong  reason  why  this  term  should  be  used.     The 


74         ESSENTIALS  OP  BACTERIOLOGY. 

blood-serum  of  immune  animals  is  simply  another  means  for 
immunization.  It  is  less  permanent  than  the  other  forms  of 
immunization,  but  it  appears  very  soon  after  the  injection,  and 
in  a  modified  form  has  a  curative  action  even  when  the  symp- 
toms of  the  infection  are  already  present  in  the  system. 

Inherited  Immunity.  An  immunity  to  disease  acquired  dur- 
ing the  lifetime  of  the  parents  is  probably  never  transmitted 
to  the  offspring,  though  the  mother  may  transmit  a  temporary 
immunity  to  the  child  in  utero  or  the  child  itself  may  have 
been  subjected  to  the  infection  at  the  same  time  with  its 
mother.     But  this  cannot  be  called  inherited. 

Theories  of  Immunity. 

Several  older  theories  need  only  to  be  mentioned,  as  they  are 
no  longer  tenable.  They  are  the  exhaustion  theory  of  Pasteur, 
the  retention  theory,  and  the  humeral  theory.  At  present 
modifications  of  MetschnikofT's  phagocytic  theory  and  Ehrlich's 
side-chain  theory  seem  the  most  plausible. 

Phagocytic  or  Cellular  Theory. — Metschnikoff  elaborated  this 
after  his  study  on  inflammation.  Phagocytosis  occurs  in 
animals  when  subjected  to  the  action  of  an  irritant.  The  leu- 
cocytes are  attracted  to  the  injured  spot  and  envelop  the  irri- 
tating substance,  be  it  bacteria  or  dead  matter.  The  theory 
given  out  at  first  was  that  if  the  leucocytes  conquer  the  bacteria, 
immunity  results;  if  the  bacteria  eat  up  the  leucocytes,  disease 
occurs. 

Modified  to  suit  other  conditions,  as,  for  instance,  the  germi- 
cidal properties  of  serum  freed  from  its  cellular  elements, 
Metschnikoffnow  states  that  at  times  phagolysis — that  is,  breaking 
up  or  solution  of  the  phagocytes — takes  place,  and  the  fluids  in 
which  these  cells  are  dissolved  become  charged  with  the  powers 
originally  present  in  the  phagocytes.  Chemotaxis  is  the  term 
applied  to  the  attraction  of  bacteria  for  the  leucocytes,  and  is 
supposed  to  be  chemical  in  its  nature.  The  phagocytic  cells 
comprise  :  (o)  The  polymorphonuclear  leucocytes  of  the  blood, 
termed  microphages,  and  (b)  a  group  called  macrophages  which 
includes  all  other  cells  having  phagocytic  properties,  such  as 
leucocytes  other  than  the  polymorphonuclears,  endothelial 
cells,  and  connective-tissue  corpuscles.    When  these  cells  are 


IMMUNITY.  75 

injured  they  set  free  their  digestive  ferments,  known  as  micro- 
cytases  and  macrocytases  respectively,  which  correspond  to  the 
alexins  of  Ehrlich. 

EhrllcWs  Side-chain  Theory.  This  derives  its  name  from  the 
fact  that  it  presents  an  analogy  to  what  happens  in  the  benzol 
ring  of  organic  chemistry  when  its  replaceable  atoms  of  hydro- 
gen are  substituted  by  "side  chains"  of  more  or  less  complex 
nature.  The  molecule  of  protoplasm  is  supposed  to  consist  of 
a  central  atom  group  provided  with  a  large  number  of  side 
chains  which  subserve  the  vital  processes  of  the  molecule  by 
combining  with  other  organic  molecules.  These  side  chains 
are  called  receptors  and  are  of  many  different  kinds  so  as  to  fit 
them  for  combination  with  many  different  varieties  of  extrane- 
ous groups.  Bacterial  toxins  contain  two  groups :  (1)  the 
haptophores,  by  which  the  toxin  molecule  can  become  joined 
to  the  cell,  and  (2)  the  toxophores,  by  virtue  of  which  it  can 
attack  the  protoplasm  after  having  been  fixed  to  it  by  the  hapto- 
phore.  If  the  attack  on  the  molecule  is  not  too  severe,  this  is 
stimulated  into  overactivity  and  throws  out  an  abnormal  num- 
ber of  receptors,  some  of  which  (the  haptins)  become  detached 
and  are  capable  of  uniting  with  free  haptophores  and  prevent- 
ing their  combination  with  the  protoplasm  of  the  molecule. 
In  other  words,  they  represent  the  antitoxin. 

Bacteriolysis  is  the  destruction  of  the  bacterial  cells  by  the 
blood-serum,  and  is  probably  effected  in  a  somewhat  different 
manner.  Antibacterial  sera  are  effective  through  the  combined 
activities  of  a  destructive  element,  the  "complement"  (alexin 
or  cytase),  and  an  "immune  body"  (amboceptor)  which  serves 
the  function  of  joining  the  complement  to  the  bacterial  mole- 
cule. These  two  bodies  differ  markedly  in  their  properties — for 
example,  the  complement  is  destroyed  at  60°  C,  while  the 
immune  body  is  very  resistant. 

It  is  not  stated  what  cells  are  the  sources  of  these  various 
anti-bodies,  but  probably  any  cell  capable  of  being  attacked  by 
a  toxin  is  also  capable  of  responding  by  the  production  of  anti- 
substances. 

Lysins.  The  substances  producing  destruction  of  bacteria 
are  called  lysins.     Normal  blood-serum  is  bacteriolytic  to  a 


76         ESSENTIALS  OF  BACTERIOLOGY. 

slight  degree,  but  during  infection  produces  lysins  specific  for 
the  germ  in  question. 

Agglutinins.  These  are  bodies  formed  in  the  blood-serum  in 
response  to  the  stimulation  of  certain  bacteria,  such  as  the 
typhoid  bacillus,  Bacillus  coli  communis,  Micrococcus  meli- 
tensis,  the  bacillus  of  dysentery,  the  cholera  spirillum,  etc. 
When  such  a  serum  is  added  to  cultures  of  the  particular 
organism  concerned,  the  bacteria  become  clumped  in  motion- 
less masses.  A  modified  form  of  agglutination  in  which  long 
strings  of  bacteria  are  formed  is  known  as  the  "  thread  "  reac- 
tion. 

Precipitins.  Animals  immunized  to  certain  bacteria  or  to 
albumins  of  different  sorts  form  bodies  which  cause  the  blood- 
serum  to  give  a  precipitate  when  added  to  cultures  of  these 
organisms  or  fluids  containing  the  specific  albumen.  The 
phenomenon  has  found  forensic  application  in  the  identifica- 
tion of  blood-stains. 


CHAPTER  XV. 

EXPERIMENTS  UPON   ANIMALS. 

The  smaller  rodents  and  birds  are  the  ones  usually  employed 
for  inoculation,  as  rabbits,  Guinea-pigs,  rats  aud  mice,  and 
pigeons,  and  chickens ;  sometimes  monkeys.  These  are  pre- 
ferred, because  easily  acted  upon  by  the  various  bacteria,  readily 
obtained,  and  not  expensive. 

The  white  mouse  is  very  prolific  and  easily  kept,  and  is  there- 
fore a  favorite  animal  for  experiment.  It  lives  well  upon  a  little 
moistened  bread.  A  small  box,  perforated  with  holes,  is  filled 
partly  with  sawdust,  and  in  this  ten  to  twelve  mice  can  be  kept. 
When  the  female  becomes  pregnant  she  should  be  removed  to 
a  glass  jar  until  the  young  have  opened  their  eyes,  because  the 
males,  which  have  not  been  raised  together,  are  apt  to  attack- 
each  other. 

Guinea-pigs.  When  Guinea-pigs  have  plenty  of  light  and 
air  they  multiply  rapidly.     Therefore  it  is  best  to  have  them  in 


EXPERIMENTS    UPON    ANIMALS.  77 

some  large  stall  or  inclosure.     They  can  be  fed  upon  all  sorts  of 
vegetables  and  grasses,  and  require  but  little  attention. 

Methods  of  Inoculation.  L  Inha lation.— Imitating  the  natural 
infection,  either  by  loading  an  atmosphere  with  the  germs  in 
question  or  by  administering  them  with  a  spray. 

II.  Tlirougk  Skin  or  Mucous  Membrane. 

III.  With  the  Food. 

Method  of  Cutaneous  Inoculation.  The  ear  of  mice  is  best 
suited  for  this  procedure.  A  small  abrasion  made  with  the 
point  of  a  lancet  or  needle,  which  has  been  dipped  in  the  virus. 
The  animal  is  then  separated  from  the  rest  and  placed  in  a 
glass  jar,  which  is  partly  filled  with  sawdust  and  covered  with 
a  piece  of  wire-gauze. 

Subcutaneous.  The  root  of  the  tail  of  mice  is  used  for  this 
purpose.  The  hair  around  the  root  of  the  tail  is  clipped  off, 
and  with  a  pair  of  scissors  a  very  small  pocket  is  made  in  the 
subcutaneous  connective  tissue,  not  wounding  the  animal  any 
more  than  absolutely  necessary,  avoiding  much  blood.  The 
material  is  placed  upon  a  platinum  needle  and  introduced  into 
the  pocket,  solid  bodies,  with  a  forceps. 

To  hold  the  mouse  still  while  the  operation  is  going  on  a 
little  cone  made  of  metal  is  used.  The  mouse  just  fits  in  here. 
There  is  a  slit  along  the  top  in  which  the  tail  can  be  fastened, 
and  thus  the  animal  is  secure  and  immobile. 

Intravenous  Injections.  Rabbits  are  very  easily  injected 
through  the  veins.     Mice  are  too  small. 

The  ear  of  the  rabbit  is  usually  taken.  It  is  first  washed  with 
1-2000  bichloride,  which  not  only  disinfects,  but  also  makes  the 
vessels  appear  more  distinct.  The  base  of  the  ear  is  compressed 
to  swell  the  veins.  Then  a  syringe,  like  the  one  used  for  the 
injection  of  "  tuberculine,"  a  Koch  syringe,  which  can  be  easily 
sterilized,  is  filled  with  the  desired  amount  of  virus  and  slowly 
injected  into  any  one  of  the  more  prominent  veins  present. 
(Fig.  43.) 

Intra-peritoneal  Injection.  This  is  used  with  Guinea-pigs 
mostly.  The  abdominal  wall  is  pinched  up  through  its  entire 
thickness,  and  the  needle  of  the  syringe  thrust  directly  through, 
so  that  it  appears  on  the  other  side,  then  the  fold  let  go,  the 
needle  withdrawn  just  far  enough  so  as  to  be  within  the  cavity. 


78 


ESSENTIALS  OF  BACTERIOLOGY. 


Inoculation  in  the  Eye.  The  anterior  chamber  and  the  cornea 
are  the  two  places  used.  The  rabbit  is  fixed  upon  a  board; 
the  eyelids  held  apart  and  head  held  still  by  an  assistant.  A 
small  cut  is  made  in  the  cornea,  a  few  drops  of  cocaine  having 
first  been  introduced  in  the  eye.  The  material  is  passed 
through  the  opening  with  a  small  forceps,  and  with  a  few  strokes 
of  a  spoon  it  is  pushed  in  the  anterior  chamber. 

For  the  cornea  a  few  scratches  made  in  the  corneal  tissue 
will  suffice  ;  the  material  is  then  gently  rubbed  in. 

Inoculation   of  the   Cerebral   Membranes.    The  skin  and 


Fig.  43. 


Manner  of  making  intravenous  injections  in  the  rabbit. 


aponeurosis  cut  through  where  the  skull  is  the  thinnest.  Then 
the  bone  carefully  trephined,  and  the  dura  exposed.  In  Babies 
inoculation,  the  syringe  containing  the  hydrophobic  virus  pierces 
the  dura  and  arachnoid,  and  the  virus  is  discharged  beneath  the 
latter. 

Intra-Tracheal.  The  bacteria  can  be  introduced  directly  into 
the  trachea,  thus  coming  in  contact  with  the  lungs. 

Intra-duodenal. — Cholera  germs  are  injected  into  the  intes- 


EXPERIMENTS    UPON    ANIMALS.  79 


tines  after  they  have  been  exposed,  by  carefully  opening  the 
abdomen.  This  is  done  in  order  to  avoid  the  action  of  the 
gastric  juice. 

Celloidin  sacs  of  small  size  are  sometimes  used  to  introduce 
living  cultures  of  bacteria  into  the  bodies  of  animals  without 
their  coming  into  direct  contact  with  the  tissues. 

Obtaining  Material  from  Infected  Animals.  The  animal 
should  be  skinned,  or  the  hairs  plucked  out,  before  it  is  washed, 
at  least  the  portion  where  the  incision  is  to  be  made.  Then  the 
entire  body  is  washed  in  sublimate.  Two  sets  of  instruments 
are  required,  one  for  coarser  and  one  for  finer  work  :  the  one 
sterilized  in  the  flame  ;  the  other,  to  prevent  being  damaged, 
heated  in  a  hot  air  oven. 

The  animal,  the  mouse  for  example,  is  stretched  upon  a  board, 
a  nail  or  pin  through  each  leg,  and  the  head  fixed  with  a  pin 
through  the  nose.  The  skin  is  dissected  awa}r  from  the  belly 
without  exposing  the  intestines.  Then  the  ribs  being  laid  bare, 
the  sternum  is  lifted  up,  and  the  pericardium  exposed.  A  pla- 
tinum needle  dipped  into  the  heart  after  the  pericardium  has 
been  slit  will  give  sufficient  material  for  starting  a  culture.  If 
the  other  organs  are  to  be  examined,  further  dissection  is  made. 
If  the  intestines  were  first  to  be  looked  at,  they  would  be  laid 
bare  first. 

In  this  manner  material  is  obtained,  and  the  results  of  inocu- 
lation noted. 

Frequent  sterilization  of  the  instruments  is  desirable. 

Koch's  Rules  in  Regard  to  Bacterial  Cause  of  Disease. 
Before  a  microbe  can  be  said  to  be  the  cause  of  a  disease,  it 
must  — 

First.  Be  found  in  the  tissue  or  secretions  of  the  animal  suf- 
fering from,  or  dead  with  the  disease. 

Second.  It  must  be  cultivated  outside  of  the  body  on  artificial 
media. 

Third.  A  culture  so  obtained  must  produce  the  disease  in 
question  when  it  is  introduced  into  the  body  of  a  healthy 
animal. 

Fourth.  The  same  germ  must  then  again  be  found  in  the 
animal  so  inoculated. 


PART  II. 
SPECIAL  BACTERIOLOGY. 


CHAPTER  I. 

NON-PATHOGENIC   BACTERIA. 

Special  Bacteriology.  Under  this  head  the  chief  character- 
istics of  individual  bacteria  will  be  detailed,  pathogenic  and  non- 
pathogenic being  the  main  divisions.  It  is  usual  to  describe  the 
non-pathogenic  first. 

Non-Pathogenic  Bacteria.  There  are  300  varieties  of  non- 
pathogenic bacteria,  and  the  list  is  continually  being  added  to. 

Bacillus  Prodigiosus.  (Ehrenberg.)  This  bacillus,  formerly 
called  a  micrococcus,  is  very  common,  and  one  of  the  first 
noticed,  because  of  the  lively  red  color  it  forms  on  vegetables 
and  starchy  substances.  "The  bleeding  host,"  miracles  being 
due  to  it. 

Form. — Short  rods,  often  in  filaments,  without  spores. 

Immobile. — Has  no  automatic  movements. 

Facultative  anaerobic,  that  is,  it  can  grow  without  air ;  but 
the  pigment  requires  oxygen  to  show  itself. 

Growth.     Gelatine.     Liquefy  rapidly. 

Colonies. — At  first  white,  round  points  with  smooth  edge 
appearing  brown  under  microscope,  but  soon  changing  to  red. 

Stab  Cultures. — The  pigment  develops  on  the  surface,  the 
growth  occurring  all  along  the  line. 

Potato  is  well  suited  to  the  growth,  the  pigment  developing 
after  twelve  hours.     Agar  and  blood  serum  growths  do  well. 

Temperature.—  Grows  best  at  25°  C. 

Varieties. — By  exposure  to  heat  of  brood-oven  during  several 
generations  the  power  to  produce  pigment  can  be  temporarily 
abolished. 

(80) 


NON-PATHOGENIC    BACTERIA 


81 


Auto- 


red 


e  Pigment. — A  pigment-forming  body  is  created  by  the 
bacillus,  and  the  action  of  oxygen  upon  it  produces  the  color. 
It  is  insoluble  in  water,  slightly  soluble  in  alcohol  and  ether ; 
acids  fade  it,  alkalies  restore  the  color.  The  pigment  resembles 
fuchsin,  presenting  the  same  metallic  lustre. 

Gases. — A  trimethylamin  odor  arises  from  all  cultures. 

Stain. — Takes  all  anilin  dyes  easily  in  the  ordinary  way. 

Bacillus  Indicus.     (Koch.)     Syn.  Micrococcus  Indicus. 

Origin. — Found  in  the  stomach  of  an  Indian  ape. 

Form. — Short  rods  with  rounded  ends.      No  spores, 
matic  movements  present ;  faadtative  ancerobin. 

Growth.     Gelatine. — Liquefy  rapidly. 

Colonies.— Round,   or  oval,  granular  margins ;   brilliant 
pigment. 

Stab  Cultures. — On  the    surface  the  pigment    shows    itself. 
Grows  well  on  other  media. 

Temperature.— Grows  best  at  35°  C. 

Action  on  Animals. — In  very  large  quantities,  if  injected  into 
the  blood,  a  severe  and  fatal  gastro- 
enteritis can  be  produced. 

Stain.—  Takes  all  dyes. 

Bacillus  Mesentericus  Vulgatus. 

The  common  potato  bacillus  of 
Fliigge. 

Habitat. — Surface  of  the   soil,  on 
potatoes,  and  in  milk. 

Form. — Small    thick    rods    with 
rounded  ends,  often  in  pairs. 

Properties. — Yery    motile  ;    pro- 
duce   abundant     spores ;      liquefy 


Fig.  44. 


gelatine ;  diastolic  action. 


Colony  of  Bacillus  Mesentericus 
Vulgatus. 

Growth.—  Rapid. 

Plate  Colonies.—  Round,  with  transparent  centre  at  first,  then 
becoming  opaque.  The  border  is  ciliated  ;  little  projections 
evenly  arranged. 

Potato.  —  A  white  covering  at  first,  which  then  changes  to  a 
rough  brown  skin  ;  the  skin  can  be  detached  in  long  threads. 

Temperature.— Spores  at  ordinary  temperatures. 
6 


82 


ESSENTIALS  OF  BACTERIOLOGY. 


Spores. — Are  very  resistant ;  are  colored  in  the  manner  de- 
scribed in  first  part  of  the  book  for  spores  in  general. 

Bacillus  Megaterium  (de  Bary). 

Origin.— Found  on  cooked  cabbage  and  garden  soil. 

Form. — Large  rods,  four  times  as  long  as  they  are  broad, 
2.5  fi.  Thick  rounded  ends.  Chains  with  ten  or  more  members 
often  formed ;  granular  cell  contents. 

Properties. — Abundant  spore  formation ;  very  slow  movement ; 
slowly  dissolves  gelatine. 

Growth.— Strongly  aerobic  ;  grows  quickly,  and  best,  at  a  tem- 
perature of  20°  C. 

Plate  Colonies.— Small,  round,  yellow  points  in  the  depth  of 
the  gelatine.     Under  microscope  irregular  masses. 

Fig.  45. 


Bacillus  Megaterium,  with  spores. 


Stab  Culture. — Funnel-shaped  from  above  downwards. 

Potato. — Thick  growth  with   abundance  of  spores. 

Bacillus  Ramosus. 

Syn.  Bac.  Mycoides  (Fliigge).     Wurzel  or  root  bacillus. 

Origin.— In  the  upper  layers  of  garden  or  farm  grounds,  and 
in  water. 

Form.— Short  rods,  with  rounded  ends,  about  three  times 
as  long  as  they  are  thick  ;  often  in  long  threads  and  chains,, 


NON-PATHOGENIC    BACTERIA.  83 

Properties. — Large,  shining,  oval  spores  ;  a  slight  movement ; 
liquefy  gelatine. 

Growth.—  At  ordinary  temperatures,  with  plentiful  supply  of 
air. 

Plate  Colonies. — Look  like  roots  of  an  old  tree  gnarled  together, 
radiating  from  a  common  centre.     On  surface  soon  liquid. 

Stab  Culture.— Soon  sl  growth  occurs  along  the  needle  track, 
and  the  whole  resembles  a  pine  tree  turned  upside  down.  The 
gelatine  then  becomes  liquid,  a  thin  skin  floating  on  top,  and 
small  flakes  lying  at  the  bottom. 

Stroke  Culture. — Feathery  resemblance  is  produced. 

Staining. — Spores  stain  readily  with  the  ordinary  spore  stain. 

Bacterium  Zopfii.  (Kurth.) 

Origin. — Intestines  of  a  fowl. 

Form. — Short  thick  rods  forming  long  threads  coiled  up, 
which  finally  break  up  into  spores,  which  were  once  thought  to 
be  micrococci. 

Properties. — Very  motile ;  does  not  dissolve  or  liquefy  gela- 
tine. 

Growth. — In  thirty  hours  abundant  growth;  cerobic;  grows 
best  at  20°  C. 

Plates. — Small  white  points  which  form  the  centre  of  a  very 
fine  netting.  With  high  power  this  netting  is  found  composed 
of  bacilli  in  coils,  like  braids  of  hair. 

Excellent  impress  or  "Klatsch"  preparations  are  obtained 
from  these  colonies. 

Staining. — Ordinary  dyes. 

Bacillus  Subtilis.     (Hay  Bacillus.)    Ehrenberg. 

Origin.—  Hay  infusions  ;  found  also  in  air,  water,  soil,  faeces, 
and  putrefying  liquids.  Very  common,  often  contaminates 
cultures. 

Form. — Large  rods,  three  times  as  long  as  broad  ;  slight 
roundness  of  ends,  transparent;  seldom  found  singly;  usually 
in  long  threads.  Flagella  are  found  on  the  ends.  Spores  of 
oval  shape,  strongly  shining,  very  resistant. 

Properties.— Yery  motile  ;  dissolves  gelatine. 

Growth. — Rapid  ;  strongly  eerobic. 

Plate. — Round,  gray  colonies,  with  depressed  white  centre. 


84        ESSENTIALS  OF  BACTERIOLOGY. 

Under  microscope  the  centre  yellow ;  the  periphery  like  a  wreath, 
with  tiny  little  rays  projecting  ;  very  characteristic. 

Potato. — A  thick  moist  skin  forms  in  twenty-four  hours. 

Staining.— Rods,  ordinary  stain,  spores,  spore  stain. 

It  is  easily  obtained  by  covering  finely  cut  hay  with  distilled 
water,  and  boiling  a  quarter  of  an  hour.  Set  aside  forty-eight 
hours.  A  thick  scum  will  show  itself  on  the  surface  composed 
of  the  subtilis  bacilli,  whose  spores  alone  have  survived  the  heat. 

Bacillus  Spinosus.    (Liideritz.) 

Called  spinosus  because  small  spine-like  processes  are  formed 
by  the  colonies. 

Origin. — In  the  juices  of  the  body  of  a  mouse  and  guinea-pig 
which  were  inoculated  with  garden  earth. 

Form.— Large  rods,  straight,  some  slightly  bent,  ends  rounded ; 
often  in  long  threads. 

Properties.  —  Large  spores,  the  bacillus  enlarging  to  allow  the 
spores  to  develop ;  very  motile  ;  gelatine  slowly  liquefied.  A 
gas  is  formed  in  the  culture  having  an  odor  like  Swiss  cheese. 

Growth. — The  growth  occurs  at  ordinary  temperatures  only 
when  the  oxygen  is  excluded.  Very  strongly  anaerobic.  Glu- 
cose added  to  the  gelatine  (1  to  2  per  cent.)  increases  the  nutri- 
tive value. 

Colonies  in  roll  cultures  and  high  stab  cultures  appear  as  little 
spheres  surrounded  by  a  zone  of  liquefied  gelatine.  In  the 
deeper  growths  thorn-like  projections  or  spines  develop  pro- 
ceeding from  a  gray-colored  centre. 

Staining.—  With  ordinary  methods.  This  bacillus,  being 
strongly  anaerobic,  must  be  cultured  with  the  usual  care  taken 
with  anaerobins. 

Some  Bacteria  found  in  Milk.  Bacillus  Acidi  Lactici. 
(Huppe.)  Belongs  to  the  same  group  as  the  Bacillus  coli  com- 
munis. 

Origin. — In  sour  milk. 

Form.— Short  thick  rods,  nearly  as  broad  as  they  are  long, 
usually  in  pairs. 

Properties.— Immotile.  Spores  large  shining  ones.  Do  not 
liquefy  gelatine.  Breaks  up  the  sugar  of  milk  into  lactic  acid 
and  carbonic  acid  gas,  the  casein  being  thereby  precipitated. 

Growth.Siow  ;  is  facultative  anaerobic.   Grows  first  at  10° C. 


NON-PATHOGENIC    BACTERIA.  85 

Plate  Colonies.— First  small  white  points,  which  soon  look  like 
porcelain,  glistening.  Under  microscope  the  surface  colonies 
resemble  leaves  spread  out. 

Stab  Culture. — A  thick  dry  crust  with  cracks  in  it  forms  on  the 
surface  after  a  couple  of  weeks. 

Attenuation. — If  cultured  through  successive  generations,  they 
lose  the  power  to  produce  fermentation.  Several  other  bacteria 
will  give  rise  to  lactic  acid  fermentation  ;  but  this  especial  one 
is  almost  constantly  found,  and  is  very  wide  spread. 

In  milk,  it  first  produces  acidity,  then  precipitation  of  casein, 
and  finally,  formation  of  gases. 

A  bacillus  described  by  Grotenfeldt,  and  called  Bacterium 
Acidi  Lactici,  forms  alcohol  in  the  milk.  It  was  found  in  milk 
in  Bavaria. 

Bacillus  Butyricus.    (Hiippe.) 

This  bacillus  causes  butyric  acid  fermentation. 

Origin. — Found  in  milk. 

Form.— Short  and  long  thin  rods  wfth  rounded  ends;  large 
oval  spores,  seldom  forming  threads. 

Properties. — Very  motile  ;  liquefies  gelatine  rapidly  ;  produces 
gases  resembling  butyric  acid  in  odor.  In  milk  it  coagulates  the 
casein,  decomposes  it,  forming  peptones  and  ammonia,  with  a 
bitter  taste,  and  butyric  acid  fermentation.  An  alkaline  reaction. 

Growth. — Quickly,  at  35°  to  40°  C,  with  oxygen.  Spons  very 
resistant. 

Colonies.  Plate.  —  Small  yellow  points  which  soon  run  together, 
becoming  indistinguishable. 

Stab  Culture.— A  small  yellow  skin  formed  on  the  surface  with 
delicate  wrinkles  ;  cloudy  masses  in  the  liquefied  portion. 

Staining. — With  ordinary  stains. 

Bacillus  Amylobacter  (Van  Tiegham) ;  or,  Clostridium  Buty- 
ricum.     (Prazmowsky.)     (Vibrion  butyrique  of  Pasteur.) 

Origin. — Found  in  putrefying  plant-infusions,  in  fossils,  and 
conifera  of  the  coal  period. 

Form. — Large,  thick  rods,  with  rounded  ends,  often  found  in 
chains.  A  large  glancing  spore  at  one  end,  the  bacillus  becoming 
spindle-shape  in  order  to  allow  the  spore  to  grow ;  hence  the 
name  Clostridium. 


86 


ESSENTIALS  OF  BACTERIOLOGY. 


Bacillus  Amylobacter. 


Fig.  46.  Properties. — Yery   motile  ;    gases    arise 

with  butyric  smell.  In  solutions  of  sugars, 
lactates  and  cellulose-containing  plants, 
and  vegetables,  it  gives  rise  to  decomposi- 
tions in  which  butyric  acid  is  often  formed. 
Casein  is  also  dissolved. 

Like  granulose,  a  watery  solution  of 
iodine  will  color  blue  some  portions  of  the 
bacillus  ;  therefore  it  has  been  called  amy- 
lobacter. 

Growth. — It  is  strongly  anaerobic,   and 
fj\      1  a   #  has  not  yet  been  satisfactorily  cultivated. 

Bacillus  Lactis  Cyanogenus.  Bacterium 
Syncyanum.  (Hiippe.) 

Origin.— Found  in  blue  milk. 
Form.—  Small  narrow  rods  about  three  times  longer  than  they 
are  broad  ;  usually  found  in  pairs.     The  ends  are  rounded. 

Properties.— They  are  very  motile  ;  do  not  liquefy  gelatine ; 
form  spores  usually  in  one  end.  A  bluish-gray  pigment  is  formed 
outside  of  the  cell,  around  the  medium.  The  less  alkaline  the 
media  the  deeper  the  color.  It  does  not  act  upon  the  milk  other- 
wise than  to  color  it  blue. 

Growth. — Grows  rapidly,  requiring  oxygen.  Colonies  on  plate. 
Depressed  centre  surrounded  by  ring  of  porcelain-like  bluish 
growth.     Dark  brown  appearance  under  microscope. 

Stab  Culture. — Grows  mainly  on  surface ;  a  nail-like  growth. 
The  surrounding  gelatine  becomes  colored  brown. 
Potato. — The  surface  covered  with  a  dirty  blue  scum. 
Attenuation. — After  prolonged  artificial  cultivation  loses  the 
power  to  produce  pigment. 

Staining. — By  ordinary  methods. 

Bacillus  Lactis  Erythrogenes.  Bacillus  of  Bed  Milk.  (Hiippe 
and  Grotenfeldt.) 

Origin. — Found  in  red  milk,  and  in  the  faeces  of  a  child. 
Form. — Short  rods,  often  in  long  filaments,  without  spores. 
Properties. — Does  not  possess  self-movement.    Forms  a  nause- 
ating odor  ;  liquefies  gelatine.   Produces  a  yellow  pigment  which 
can  be  seen  in  the  dark,  and  a  red  pigment  in  alkaline  media, 


NON-PATHOGENIC    BACTERIA.  87 

away  from  the  light.  In  milk  it  produces  the  yellow  cream  on 
top  of  the  blood-red  serum,  or,  fluid  in  the  centre,  and  at  the 
bottom  the  precipitated  casein. 

Growth. — Grows  rapidly  in  bouillon  and  on  potatoes ;  slower 
on  the  other  media;  Plates.  A  cup-like  depression  in  the  centre 
of  the  colony,  with  a  pink  coloration  around  it,  the  colony  itself 
being  slightly  yellow. 

Stab  Culture. — The  growth  mostly  on  surface.  The  gelatine 
afterwards  colored  red  and  liquefied. 

Potato. — A  golden  yellow  pigment  formed  at  37°  C,  after  six 
days. 

Examination  of  Milk  in  Stained  Specimen.  A  drop  of  milk 
diluted  with  a  drop  of  distilled  water  is  dried  on  the  cover-glass 
and  fixed  by  heat.  Chloroform  methyl  blue,  prepared  by  mix- 
ing 12  to  15  drops  of  saturated  alcoholic  solution  of  methyl  blue 
with  3  or  4  c.c.  of  chloroform,  is  used  for  staining.  The  chlo- 
roform is  then  evaporated  by  exposing  the  specimen  for  a  few 
minutes  to  the  air.    Bacteria  blue ;  rest  of  field  unstained. 

Another  method  is  to  mix  a  drop  of  milk  with  two  or  three 
drops  of  a  1  per  cent,  solution  of  sodium  carbonate  on  a  cover- 
glass.  Saponification  of  the  fat  occurs  on  heating  the  mixture 
to  evaporation.  The  preparation  is  then  stained  in  the  ordinary 
manner. 

Some  Non-Pathogenic  Bacteria  found  in  Water.  The  bacteria 
found  here  are  very  often  given  to  producing  pigments  or  phos- 
phorescence, and  are  in  great  number.  The  more  common  ones 
only  will  be  described. 

Bacillus  Violaceus. 

Origin.  — Water. 

Form.— A  slender  rod  with  rounded  ends,  three  times  as  long 
as  it  is  broad,  often  in  threads  ;  middle-sized  spores. 

Properties. — Very  motile  ;  forms  a  violet-blue  pigment,  which 
is  soluble  in  alcohol,  and  depends  upon  oxygen  for  its  growth. 
Rapidly  liquefies  gelatine,  but  not  agar. 

Growth.— Grows  fairly  quick,  is  facultative  anaerobic. 

Cultures  on  Plate.— At  first  the  colonies  look  like  inclosed  air- 
bubbles.  Low  power  shows  irregular  masses,  with  a  centre 
containing  the  pigment  and  a  hairy-like  periphery. 


growth 


—Terr  fine  little  rods ;  no 
—Motile  5  fbnns 

gelatine. 
Rapid  onhrafc  ordinary 


the  periphery 
the  needle  thrust ;  the 


MOX-PATHOOEfrlC    BACTERIA,  $$ 

Plate*.— Bound  colonies,  cup-shaped  depressions,  the  solid 
geb:ine  that  remains  becoming  colored  witb  greenish-yellow 

Stab  Culture.— ()n  the  surface,  air-bobble  depressions;  toe 
white  colonies  in  the  bottom  of  these  depressions,  and  the  solid 
gelatine  aroond  the  inoculation  shining  with  the  fluorescence. 

Phosphorescent  Bacteria,  Six  varieties  of  phosphorescent 
bacteria  bare  been  described ;  they  are  found  usually  in  sea* 
water,  or  upon  objects  firing  in  the  sea, 

Bacillus  Photphoreseens  Indieus.    (Fischer,) 

Origin.— Tropical  waters. 

Form.— Thick  rods,  with  rounded  ends,  sometimes  forming 
bag  Uueada 

Properties.— Very  motile ;  liquefying  gelatine  at  a  tempera* 
tare  of  2KP  to  Sf/"-  C,  with  oxygen  and  a  little  moisture,  and  in 
the  dark,  a  peculiar  electric-bine  light  develops  a  phosphores- 
cence. 

Growth.— Slowly ;  must  bare  oxygen ;  does  not  grow  under 
1CPC,  ororerSCPC, 

Plates,— Little  round,  gray  points,  which  under  low  power 
appear  as  green  colonies  with  reddish  tinge  around  them. 
Cooked  fish,  when  smeared  upon  the  surface  with  a  little  of  the 
culture,  show  the  phosphorescence  most  marked.  Grows  well 
on  potatoes  aid  Wood-serum. 

Bacmusl^jsphcresceiis  Indigents    (Fischer,) 

Origin.—  Waters  in  the  northern  part  of  Germany,  It  differs 
from  the  Indian  bacillus,  in  that  it  grows  at  a  temperature  of 
IP  C,  and  does  not  develop  upon  potatoes  or  blood-serum, 

BaciUiuPbospbcmseeiuGetidiiA    (Fdrster,) 

Origin — Surfaces  of  salt-water  fish. 

Form.— Short,  thick  rods,  looking  oral  sometimes ;  zoogkea 
arc  often  formed. 

Properties.— Motile ;  does  not  liquefy  gelatine ;  a  beautiful 
phosphorescence  from  the  surface  of  fish;  it  can  be  photographed 
by  its  own  light 

Colonies.— Grows  best  between  (P  and  2<P  C. ;  grows  slowly, 
and  mostly  on  the  surface.  The  material  must  contain  salt. 
A  bouillon  made  with  sea-water,  or  3  to  4  per  cent,  common 


90         ESSENTIALS  OF  BACTERIOLOGY. 

salt  will  suffice.     The  colonies  appear  as  those  of  the  Phospho- 
rescens  Indicus. 

Fresh  herring  laid  between  two  plates  will  often  show  phos- 
phorescence in  twent}r-four  hours. 

The  other  three  varieties  require  glucose  in  the  culture  before 
they  give  out  any  glow.  They  are  Bacterium  Pjlugeri,  Bact. 
Fischeri,  and  Bact.  Baltlcum.     They  do  not  dissolve  gelatine. 

Several  very  indistinct  species,  found  in  waters  from  factories 
and  in  some  of  the  mineral  waters,  deserve  yet  to  be  men- 
tioned. They  have  been  given  various  names  by  observers ; 
almost  a  new  classification  created.  Such  are  the  crenothrix, 
cladothrix,  and  beggiatoa,  which  belong  to  the  "  higher  bacteria." 

Crenothrix  Kuhniana.  (Kabenhorst.)  Long  filaments  joined 
at  one  end  ;  little  rod-like  bodies  form  in  the  filaments  ;  and 
these  break  up  into  spores. 

Zoogloea  are  also  formed  by  means  of  spores  ;  and  these  can 
become  so  thick  as  to  plug  up  pipes  and  carriers  of  water. 
They  are  not  injurious  to  health. 

Cladothrix  Dichotoma.  (Cohn.)  Very  common  in  dirty 
waters.  The  filaments  branch  out  at  acute  angles,  otherwise 
resembling  the  crenothrix ;  accumulations  of  ochre-colored 
slime,  consisting  of  filaments  of  this  organism,  are  found  in' 
springs  and  streams. 

Leptothrix  Buccalis.  In  the  mouth  long  filaments  or  threads 
resembling  bacteria  are  commonly  found.  At  one  end  are  seen 
numerous  cocci-like  bodies,  which  some  regard  as  spores.  A 
variety  of  this,  or  a  nearly  allied  organism,  is  the  most  frequent 
cause  of  noma  or  gangrenous  stomatitis. 

With  iodin  the  leptothrix  is  colored  yellow.  At  one  time  it 
was  considered  the  cause  of  "tartar"  on  the  teeth,  and  often  it 
fills  the  crypts  of  the  tonsils,  forming  there  small  masses  which 
are  difficult  to  remove.  Miller  distinguishes  three  varieties — 
Leptothrix  buccalis  innominata,  maxima,  and  gigantea. 

Beggiatoa  Alba.  (Vancher.)  The  most  common  of  this 
species.  The  distinction  between  this  and  the  preceding  species 
lies  in  the  presence  of  sulphur  granules  contained  in  the  struc- 
ture, and  hence  they  are  often  found  where  sulphur  or  sulphides 
exist ;  but  where  the  remains  of  organic  life  are  decomposing 
they  can  also  be  found. 


NON-PATHOGENIC     BACTERIA.  91 

Several  large  spirilla  and  vibrio  live  in  bog  and  rain-water, 
but  our  space  does  not  suffice  to  describe  them. 

Micro-organisms  found  in  Urine.  When  freshly  passed,  urine 
of  a  normal  state  contains  no  bacteria.  By  contact  with  the  air 
and  the  urinary  passages  exposed  to  air,  a  great  number  of  yeast 
moulds  and  bacteria  soon  accumulate  in  the  fluid.  Bacteria 
also  enter  urine  through  the  blood  and  during  its  secretion. 

A  number  of  bacteria  have  the  property  of  converting  urea 
into  carbonate  of  ammonia. 

The  urine  should  be  centrifuged  and  the  deposit  then  exam- 
ined. The  drying  and  fixing  must  proceed  very  slowly,  since 
otherwise  crystals  of  salts  will  be  precipitated  and  mar  the 
specimen. 

Bacterium  TJreae. 

Origin. — Decomposed  ammoniacal  urine. 

Form. — Thick,  little  rods,  with  round  ends  one-half  as  thick 
as  they  are  long. 

Properties.—  Does  not  dissolve  gelatine ;  changes  urea  into 
carbonate  of  ammonia. 

Growth.— At  ordinary  temperatures,  very  slowly.  In  two  days 
on  gelatine  very  minute  points,  which  in  ten  days  have  the  size 
of  a  cent.     The  colonies  grow  in  concentric  layers. 

Micrococcus  Urese.     (Pasteur  and  Van  Tiegham.) 

Origin. — Decomposed  urine  and  in  the  air. 

Form. — Cocci,  diplococci,  and  streptococci. 

Properties.—  Decomposes  urea  into  carbonate  of  ammonia  ; 
does  not  liquefy  gelatine. 

Growth.— Grows  rapidly,  needing  oxygen;  can  remain  sta- 
tionary below  0°  C. ;  growing  again,  when  a  higher  temperature 
\s  reached. 

Colonies  on  Plate.— On.  the  surface  like  a  drop  of  wax. 

Stab  Cultures.— Looks  like  a  very  delicate  thread  along  the 
needle  thrust. 

Other  bacteria  are  found  in  urine  in  various  pathologic  pro- 
cesses, such  as  tubercle  bacilli,  typhoid  bacilli,  gonococci,  and 
other  pyogenic  organisms. 

The  Urobacillus  liquefaciens,  found  by  Schnitzler  and  Kro- 
gius  in  cystitis,  is  supposed  to  stand  in  close  relationship  to  this 
disease. 


92         ESSENTIALS  OF  BACTERIOLOGY. 

Spirillum.    Spirillum  Rubrum.    (Esmarch.) 

Origin. — Body  of  a  mouse  dead  with  septicaemia. 

Form.  —  Spirals  of  variable  length,  long  joints,  flagella  on  each 
end  ;  no  spores. 

Properties. — Does  not  liquefy  gelatine  ;  very  motile  ;  produces 
a  wine-red  pigment,  which  develops  only  by  absence  of  oxygen. 

Growth. — Can  grow  with  oxygen,  but  is  then  colorless  ;  grows 
very  slowly  ;  ten  to  twelve  days  before  any  sign  ;  grows  best  at 
37°  C. 

Gelatine  Boll  Cultures.  —Small,  round  ;  first  gray,  then  wine- 
red  colonies. 

.Stab  Cultures. — A  red-colored  growth  along  the  whole  line  ;  it 
is  deepest  below,  getting  paler  as  it  approaches  the  surface. 

Spirillum  Concentricum.    (Kitasato.) 

Origin. — Decomposed  blood. 

Form. — Short  spirals,  two  to  three  turns,  with  pointed  ends  ; 
it  has  flagella  on  the  ends. 

Properties. — Very  motile  ;  does  not  liquefy  gelatine. 

Growth. — Very  slow  ;  mostly  on  the  surface  ;  best  at  ordinary 
temperatures. 

Plates. — A  growth  of  rings  concentrically  arranged,  every 
alternate  one  being  transparent ;  the  furthest  one  from  the 
centre  possessing  small  projections. 

Stab  Cultures. — Growth  mostly  on  the  surface. 

Sarciua.  Cocci  in  cubes  or  packets  of  colonies.  A  great 
number  have  been  isolated  ;  many  producing  very  beautiful 
pigments.     The  majority  of  them  found  in  the  air. 

Sarcina  Lutea.    ( Seh roter. ) 

Origin. — Air. 

Form. — Very  large  cocci  in  pairs  ;  tetrads  and  groups  of 
tetrads. 

Properties. — Liquefies  gelatine  slowly  ;  produces  sulphur-yel- 
low pigment. 

Growth. — Slowly  ;  at  various  temperatures  ;  strongly  aerobic. 

Plates. — Small,  round,  yellow  colonies. 

Stab  Cultures.— -Grows  more  rapidly,  the  growth  being  nearly 
all  on  the  surface,  a  few  separated  colonies  following  the  needle 


NON-PATHOGENIC    BACTERIA  93 

thrust  for  a  short  distance.      Agar,  a  very  beautiful  yellow, 
along  the  stroked  surface. 

Sarcina  Aurantica. — Flava,  rosea,  and  alba  are  some  of  the 
other  varieties.     Many  are  obtained  from  beer. 

Sarcina  Ventriculi.    (Goodsir.)    (Fig.  47.) 

Origin. — Stomach  of  man  and  animals. 

Form. — Colorless,  oval  cocci,  in  groups  of  eight  and  packets 
of  eight. 

Fig.  47. 


Sarcina  ventriculi  from  stomach-contents ;  X  530.    (Van  Valzah  and  Nisbet.) 

Properties.  —Does  not  liquefy  gelatine  ;  shows  the  reaction  of 
cellulose  to  iodine. 

Growth.—  Rapid.  At  end  of  thirty-six  hours,  round,  yellow 
colonies,  from  which  colorless  cocci  and  cubes  are  obtained. 

Habitat.— They  are  found  in  many  diseases  of  the  stomach, 
especially  when  dilatation  exists.  Also  normally;  increased 
when  fermentation  occurs. 

Boas-Oppler  Bacillus,  also  known  as  the  Bacillus  geniculatus. 
Owing  to  the  faculty  possessed  by  this  organism  of  growing  in 
the  presence  of  amounts  of  lactic  acid  sufficient  to  check  the 
development  of  all  other  lactic-acid  formers,  it  usually  pre- 
dominates in  stomach-contents  containing  large  amounts  of 
this  substance.  The  parent  type  is  composed  of  short  rods, 
but  in  the  presence  of  considerable  amounts  of  lactic  acid  these 


94         ESSENTIALS  OF  BACTERIOLOGY. 

change  to  a  longer  form  which  occurs  singly  or  in  long  chains. 
It  is  stained  brown  by  Gram's  iodine  solution.  The  bacillus 
affords  confirmatory  evidence  of  the  presence  of  a  new  growth, 
though  it  may  occur  in  benign  conditions. 


CHAPTER  II. 

PATHOGENIC  BACTERIA. 

"We  have  divided  this  part  into  two  portions. 

I.  Those  bacteria  which  are  pathogenic  for  man  and  other 
animals. 

II.  Those  bacteria  which  do  not  affect  man,  but  are  patho- 
genic for*the  lower  animals. 

Here  again  it  will  only  be  possible  to  give  the  more  impor- 
tant bacteria  ;  there  are  many  diseases  in  which  micro-organisms 
have  been  found,  but  they  have  not  yet  been  proven  as  causa- 
tive of  the  disease,  and  have  also  been  found  in  other  diseases. 
We  cannot  treat  of  them  here. 

Bacillus  Anthracis.  {Bayer  and  Davaine.) — Kayer  and  Da- 
vaine,  in  1850,  first  described  this  bacillus;  but  Pasteur,  and 
later  Koch,  gave  it  the  importance  it  now  has. 

Synonyms  —  Bactericie  du  charbon  (Fr.),  Milzbrand  bacillus 
(German)  ;  bacillus  of  splenic  fever,  or  malignant  pustule. 

Origin. — In  blood  of  anthrax-suffering  animals. 

Form.—  "Rods  of  variable  length,  nearly  the  size  of  a  human 
blood-corpuscle,  broad  cup-shaped  ends;  in  bouillon  cultures, 
long  threads  are  formed,  with  large  oval  spores. 

Properties. — Liquefies  gelatine ;  immotile ;  the  spores  are  very 
resisting,  living  twenty  years,  and  resist  boiling  for  five  minutes. 

Growth.— Grows  rapidly,  between  12°  C.  and  45°  C,  and  re- 
quires plenty  of  oxygen,  but  may  be  classed  as  a  facultative 
anaerobe ;  grows  well  in  all  media. 

Plates  of  Gelatine. — Colonies  develop  in  two  days,  white  shiny 
spots,  which  appear  under  microscope  as  slightly  yellowish 
granular  twisted  balls,  like  a  ball  of  yarn ;  each  separate  string 


PATHOGENIC    BACTERIA.  95 

or  hair,  if  looked  at  under  high  power,  being  composed  of  bac- 
teria in  line. 

Stab  Cultures.— A  white  growth  with  thorn-like  processes  along 
the  needle-track ;  later  on,  gelatine  liquefied,  and  flaky  masses 
at  the  bottom. 

Potato.— A  dry  creamy  layer,  and  when  placed  in  brood-oven, 
rich  in  spores. 

Varieties.  Asporogenic.—'By  cultivation  in  gelatine,  contain- 
ing 1  to  1000  ac. carbolic,  a  variety  develop  that  cannot  produce 
spores.     Also  involution  forms,  differing  from  the  usual  type. 

Fig.  48. 


Anthrax  bacilli  in  human  blood  (fuchsin  staining),  Zeiss  1-12  oil  immersion. 
No.  4  ocular  taken  from  Vierordt. 

Staining. — They  readily  take  all  the  aniline  dyes  with  the 
ordinary  methods.  To  bring  out  the  cup-shaped  concave  ex- 
tremities, a  very  weak  watery  solution  of  methylin  blue  is  best. 

Spores  are  stained  by  the  usual  method.  When  several  bacilli 
are  joined  together,  the  place  of  their  joining  looks  like  a  spore 
because  of  the  hollowed  ends.  The  double  staining  will  develop 
the  difference. 

Sections  of  tissue  are  stained  according  to  the  ordinary 
methods,  taking  Gramas  method  very  nicely. 

Pathogenesis. — When  mice  are  inoculated  with  anthrax  mate- 


96 


ESSENTIALS  OF  BACTERIOLOGY. 


rial  through  a  wound  in  the  skin,  they  die  in  twenty-four  hours 
from  an  active  septicaemia,  the  point  of  inoculation  remaining 
unchanged,  tfhe  following  appearances  then  present  them- 
selves : — 

Peritoneum.— Covered  with  a  gelatinous  exudate. 

Spleen. — Yery  much  swollen,  dark  red,  and  friable. 

Liver. —Parenchymatous  degeneration. 


FiG.  49. 


Fig.  50. 


*& 


Stab  Cultures  of  Anthrax  in  Gelatine. 


Blood. — Dark  red.  The  bacilli  are  found  wherever  the  capil- 
laries are  spread  out,  in  the  spleen,  liver,  intestinal  villi,  and 
glomeruli  of  kidney,  and  in  the  blood  itself.  Only  when  the 
capillaries  burst  are  they  found  in  the  tubules  of  the  kidney. 

Mode  of  Entrance. — The  bacilli  can  be  infialed,  and  then  a 
pneumonia  is  caused,  the  pulmonary  cells  containing  the  bacilli ; 
when  the  spores  are  inhaled,  a  general  iufection  occurs. 


PATHOGENIC    BACTERIA.  97 

Feeding. — The  cattle  graze  upon  the  meadows,  where  the 
blood  of  anthrax  animals  has  flowed  and  become  dried,  the 
spores  remaining,  which  then  mix  with  the  grass  and  so  enter 
the  alimentary  tract ;  here  they  then  cause  the  intestinal  form 
of  the  disease,  ulcerating  through  the  villi. 

Local  Infection. — In  man  usually  only  a  local  action  occurs  ;  by 
reason  of  his  occupation — wool-sorter,  cattle-driver,  etc.,  he 
obtains  a  small  wound  on  the  hand,  and  local  gangrene  and 
necrosis  set  in. 

Pneumonia  by  inhalation  and  intestinal  infection  also  occurs 
in  man. 

Susceptibility  of  Animals. — Dogs,  birds,  and  cold-blooded  ani- 
mals affected  the  least ;  while  mice,  sheep,  and  guinea-pigs 
quickly  and  surely. 

Products  of  Anthrax  Bacilli. — A  basic  ptomaine  has  not  been 
found,  but  a  toxalbumen  or  proteid,  called  anthraxin,  has  been 
obtained.  A  certain  amount  of  acid  is  produced  by  the  virulent 
form,  alkali  by  the  weak. 

Attenuation  and  Immunity. — Cultures  left  several  days  at  a 
temperature  between  40°  and  42°  C.  soon  become  innocuous,  and 
when  injected  into  animals  protect  them  against  the  virulent 
form. 

The  lymph  obtained  from  lymph-sac  of  a  frog  destroys  the 
virulence  of  anthrax  bacilli  and  spores  temporarily. 

Hankin  obtained  an  alexin  from  the  blood  and  spleen  of  rats, 
they  being  naturally  immune.  It  destroyed  the  anthrax  bacilli 
in  vitro,  and  used  by  injection  in  susceptible  animals  made 
them  immune.     It  is  insoluble  in  alcohol  or  water. 

Protective  Vaccination. — Animals  have  been  rendered  immune 
by  various  ways — by  inoculation  of  successive  attenuated  cul- 
tures ;  also  with  sterilized  cultures — that  is,  cultures  containing 
no  bacilli,  and  with  cultures  of  other  bacteria. 

Habitat. — The  anthrax  disease  seems  confined  to  certain  dis- 
tricts in  Siberia,  Bavaria,  and  Auvergne,  and  mainly  during  the 
summer  months. 

The  bacillus  has  never  been  found  free  in  nature. 

Bacillus  Tuberculosis.    (Koch.) 

This  very  important  bacillus  was  first  described,  demonstrated, 


98         ESSENTIALS  OF  BACTERIOLOGY. 

and  cultivated  by  Koch,  who  made  his  investigations  public  on 
the  24th  of  March,  before  the  Physiological  Society  of  Berlin, 
in  the  year  1882. 

Origin. — In  various  tubercular  products  of  man  and  other 
animals. 

Form. — Very  slender  rods,  nearly  straight,  about  one-quarter 

Fig.  51. 


/    > 
2~     * 


*     »     ^ 

Tubercle  bacilli  in  sputum,  carbol-fuchsin,  and  methylin  blue.    Zeiss  1:12  oil 
immersion. 

the  size  of  a  red  corpuscle's  diameter,  their  ends  rounded,  usu- 
ally solitary,  often,  however,  lying  in  pairs  in  such  a  manner  as 
to  form  an  acute  angle.  Sometimes  they  are  S  -shaped.  In 
colored  preparations  little  oval  spaces  are  seen  in  the  rod,  which 
resemble  spores  ;  but  the  question  of  the  existence  of  spores  is 
still  undecided. 

Properties. — Does  not  possess  self-movement. 

Growth.—  Requires  special  media  for  its  growth,  and  a  temper- 
ature varying  but  slightly  from  37.5°  C.  It  grows  slowly,  de- 
veloping first  after  ten  days,  reaching  its  maximum  in  three 
weeks.  It  is  facultative  anaerobic.  On  gelatine  it  does  not 
form  a  growth. 


PATHOGENIC    BACTERIA.  99 

Colonies  on  Blood  Serum. — Koch  first  used  blood  serum  for 
culture  ground,  and  obtained  thereon  very  good  growths.  Test- 
tubes  with  stroke  culture  were  placed  in  the  brood  oven  at  37°  C. 
for  ten  to  fourteen  days,  when  small  glistening  white  points  ap- 
peared which  then  coalesced  to  form  a  dry,  white,  scale-like 
growth.  Under  microscope  composed  of  many  fine  lines  con- 
taining the  tubercle  bacillus. 

Glycerine  Agar.— By  adding  four  to  six  per  cent,  glycerine  to 
ordinary  agar-peptone  medium,  Nocard  and  Koux  obtained  a- 
culture  ground  upon  which  tubercle  bacilli  grew  much  better 
than  upon  blood  serum.     This  is  now  almost  exclusively  used. 

Stroke  cultures  are  here  used  as  with  blood  serum.  They  are 
placed  in  brood-oven  after  inoculation,  and  remain  there  about 
ten  days,  at  a  temperature  of  37°  C.  p10>  52 

The  cotton  plugs  of  the  tubes  are 
covered  with  rubber  caps,  the  cotton 
first  having  been  passed  through  the 
flame,  and  moistened  with  a  few  drops 
of  sublimate  solution.  The  rubber 
cap  prevents  the  evaporation  of  the 
water  of  condensation  which  always 
forms,  and  keeps  the  culture  from  dry- 
ing up. 

The  growth  Which  OCCUrs  resembles        Tubercle  bacilli    in   human 

the  rusas  of  the  stomach,  and  some-  liver  500  X-  (Frankei  and 
times  looks  like  crumbs  of  bread  moist- 
ened. The  impression  or"Klatsch"  preparation  shows  under 
the  microscope  a  thick  curled-up  centre  around  which  threads 
are  wound  in  all  directions.  And  these  fine  lines  show  the 
bacilli  in  profusion. 

Potato.— It  can  be  cultivated  on  slices  of  potato  which  are 
placed  in  air-tight  test-tubes. 

Bouillon.  —  Bouillon  containing  four  per  cent,  glycerine  is  a 
verv  good  nurture  ground. 

Varieties. — Branching  and  other  aberrant  forms  are  not  rare, 
and  the  tendency  now  is  to  class  the  organism  with  the  "  higher 
bacteria."     Other  acid-fast  bacilli  exhibit  similar  types  and  it  is 


100 


ESSENTIALS  OF  BACTERIOLOGY. 


possible  that  the  bacillary  parasitic  form  is  only  one  stage  in 
the  life  history  of  the  organism. 

Little  granules  arranged  like  streptococci,  which  take  the 
characteristic  stain,  and  look  as  if  the  protoplasm  had  been 
destroyed  that  enclosed  them,  are  frequently  found  in  sputum. 


Fig.  53. 


Klatsch  preparation. 

Bovine  tubercle-bacilli  are  about  one-third  smaller  than  human 
tubercle  bacilli. 

Staining.—  The  tubercle  bacilli  require  special  methods  to 
stain  them,  and  a  great  number  have  been  introduced.  They 
are  stained  with  great  difficulty :  but  once  stained,  they  are 
very  resistant  to  decolorizing  agents.  Upon  these  facts  all  the 
methods  are  founded. 


PATHOGENIC    BACTERIA.  101 

It  will  only  be  necessary  to  describe  those  methods  principally 
in  use ;  and  as  the  examination  of  sputum  for  bacilli  is  of  so 
frequent  an  occurrence,  and  so  necessary,  it  is  well  to  detail  in 
particular  the  method  of  staining. 

Starting  with  the  sputum,  we  search  for  little  clumps  or  rolled- 
up  masses  ;  if  these  are  not  present,  the  most  solid  portions  of  the 
mucus  are  brought  with  forceps  upon  a  clean  cover-glass  ;  very 
little  suffices.  With  another  cover-glass  it  is  pressed  and  spread 
out  evenly  ;  drawing  one  glass  over  the  other,  we  obtain  two 
specimens,  and  these  put  aside  or  held  high  over  the  flame  until 
dry. 

If  we  desire  to  examine  the  specimen  quickly,  or  make  a 
hurried  diagnosis,  we  use  the  rapid  method,  with  hot  solutions  ; 

Fig.  54. 


Growth  on  Agar. 

otherwise  we  let  it  stay,  in  cold  solution  until  the  next  mornin<* 
the  advantages  of  which  will  be  later  on  described. 

Tlie  Eapid  Method.— (B.  Frankel's  method  modified  by  Gab- 
bet.)  The  principle  is  to  combine  with  the  contrast  stain  the 
decolorizing  agent ;  but  the  preparations  are  not  permanent ; 
the  method,  however,  is  very  useful. 

Two  solutions  are  required :  one  of  Ziehl's  carbol-fuchsin ; 
the  other  Gabbet's  acid  methylin  blue.  (See  No.  X.,  on 
page  34. ) 

The  cover-glass  containing  the  dried  sputum  is  passed  three 
times  through  the  flame,  as  described  in  the  general  directions. 
It  is  then  placed  in  the  carbol-fuchsin  solution  five  minutes  (cold), 
or  two  minutes  in  the  hot,  immediately  then  transferred  to  the 


102  ESSENTIALS    OP    BACTERIOLOGY. 

second  solution,  the  acid  blue,  where  it  remains  one  minute, 
then  washing  in  water.  The  preparation  is  dried  between 
filter-paper,  and  mounted  best  first  in  water.  Examined  with 
oil-immersion. 

A  somewhat  longer,  but  preferable,  method  is  to  decolorize 
the  carbol-fuchsin  with  weaker  acid.  The  smear  is  treated  with 
5  per  cent,  nitric  or  10  per  cent,  sulphuric  acid  until,  after 
washing  with  water,  a  bright  pink  remains.  The  excess  of 
color  is  then  washed  out  with  95  per  cent,  alcohol  until  no 
further  color  is  imparted  to  the  alcohol  and  the  smear  is  a 
pinkish  gray.  The  preparation  is  then  washed  writh  water  and 
counterstained  with  aqueous  methylin-blue  for  ten  to  thirty 
seconds.  A  mechanical  stage  is  of  great  assistance  in  the 
search  for  the  bacilli,  as  it  permits  every  portion  of  the  prepa- 
ration to  be  inspected  systematically. 

In  urine,  owing  to  the  almost  inevitable  contamination  with 
the  smegma  bacillus,  special  methods  are  necessary  to  avoid 
error.  The  preparation  may  be  left  in  97  per  cent,  alcohol  for 
eight  hours,  when  the  smegma  bacillus  will  have  become  de- 
colorized, or  Pappenheim's  method  may  be  used :  (1)  Smear 
and  fix  as  usual ;  (2)  stain  with  hot  carbol-fuchsin  for  two 
minutes,  pour  off  the  surplus  dye  without  washing  ;  (3)  counter- 
stain  and  decolorize  by  pouring  five  times  over  the  preparation 
the  following  solution:  A  1  per  cent,  alcoholic  solution  of 
corallin  is  saturated  with  methylin-blue  and  20  parts  of  gly- 
cerine added.  Wash  in  water,  dry  with  blotting-paper,  then  in 
the  air,  and  examine.  The  tubercle  bacilli  are  stained  red, 
smegma  bacilli,  blue. 

The  bacillus  of  leprosy  resembles  the  tubercle  bacillus  in  its 
staining  properties,  but  gives  up  the  carbol-fuchsin  more  easily 
and  is  usually  decolorized  by  the  acid  and  alcohol.  It  is  colored 
blue  by  Pappenheim's  method. 

Acid-fast  bacilli  have  also  been  obtained  from  timothy  grass, 
butter,  milk,  manure,  and  the  surfaces  of  animal  bodies,  but 
differ  from  the  tubercle  bacillus  in  cultural  characteristics. 

Slow  Method. — The  stain  may  also  be  used  without  heating, 
though  in  this  case  a  much  longer  time  is  required  before  the 
bacilli  take  up  the  stain.     The  preparation  is  left  in  a  small 


PATHOGENIC    BACTERIA.  103 

dish  or  beaker  full  of  carbol-fuchsin  for  eight -to  ten  hours,  and 
then  decolorized  and  counterstained  in  the  usual  way.  The 
method  is  less  liable  to  produce  artefacts  than  the  quick 
method,  but  is  not  much  used  on  account  of  the  time  it  takes. 

BiederVs  Method  of  Collecting  Bacilli,  when  the  bacilli  are 
very  few  in  a  great  quantity  of  fluid,  as  urine,  pus,  abundant 
mucus,  etc.,  Biedert  advises  to  mix  15  c.cm.  of  the  fluid 
with  75  to  100  c.cm.  water  and  a  few  drops  of  potassium  or 
sodium  hydrate,  then  boiling  until  the  solution  is  quite  thin.  It 
is  placed  in  a  conical  glass  for  two  days,  and  bacilli  with  other 
morphological  elements  sink  to  the  bottom  of  the  glass ;  when 
the  supernatant  liquid  is  decanted,  the  residue  can  be  easily 
examined.  In  this  way  bacilli  were  found  that  had  eluded 
detection  examined  in  the  ordinary  manner. 

The  centrifugal  machine  is  used  either  in  connection  with 
Biedert's  sediment  method  or  without,  to  obtain  the  solids  sus- 
pended in  urine  or  serum. 

When  the  bacilli  are  so  few  in  number  in  sputum  or  urine  as 
to  make  their  detection  difficult,  and  also  when  doubt  exists  as 
to  the  identity  of  acid-fast  bacilli  found,  several  guinea-pigs 
should  be  injected  in  the  groin  and  smears  and  sections  made 
from  the  enlarged  glands  resulting. 

Carbolic  Acid  to  Sediment  Sputum. — Pure  carbolic  acid  added 
to  sputum  (about  1  part  of  the  acid  to  6  parts  of  sputum)  will 
in  a  few  hours  produce  a  coagulation  and  allow  the  sputum  to 
be  spread  evenly  on  the  cover-glass,  showing  greater  collections 
of  bacilli. 

Without  cover-glass. — Sputum  can  be  spread  and  stained  on 
the  glass  slide  without  the  use  of  a  cover-glass,  the  oil  of  cedar 
being  placed  directly  on  the  stained  sputum,  and  the  oil  immer- 
sion lens  dipping  into  it.  It  is  a  rapid  and  cheap  way  ;  and 
when  a  given  case  is  to  be  studied  daily  the  method  is  useful. 

Pure  Cultures  from  Sputum. — Kitasato  recommends  the  tho- 
rough washing,  changing  the  water  ten  times,  of  the  small  masses 
found  in  the  sputum  of  tubercular  persons.  When  such  speci- 
mens are  examined  they  show  tubercle  bacilli  alone,  and  when 
inoculated  in  agar  give  rise  to  pure  cultures. 

Staining  Bacillus  Tuberculosis  in  Tissue  (sections). — The  general 


104  ESSENTIALS    OF    BACTERIOLOGY. 

method  of  Gram  can  be  used,  but  the  better  way  is  to  use  the 
following : — 

Carbol-fuchsin,  15  to  30  minutes. 
5  per  cent,  sulphuric  acid,  1  minute. 
Alcohol,  until  a  light-red  tinge  appears. 
Weak  methylin  blue,  3  to  5  minutes. 
Alcohol,  for  a  few  seconds. 
Oil  of  cloves,  until  cleared. 
Canada  balsam,  to  mount  in. 
Instead  of  carbol-fuchsin,  alcoholic  solution  qffuchsin  or  aniline 
water  fuchsin  can  be  used,  but  the  sections  must  remain  in  the 
stain  over  night. 

Hardened  sputum  and  sectioning. — Sputum  can  be  hardened  l»v 
placing  it  in  98  per  cent,  alcohol.  Thin  sections  can  be  obtained 
by  imbedding  the  hardened  sputum  in  collodion.  The  sections 
are  then  stained  as  ordinary  tissue  sections. 

To  preserve  sputum. — Sputum  can  be  preserved  for  future  use 
by  placing  it  in  alcohol,  where  it  can  be  kept  for  months.  Cover- 
glass  preparations  can  then  be  made  by  softening  the  coagula 
with  a  small  amount  of  liquor  potassa. 

The  resisting  action  of  the  bacillus  to  acids  is  supposed  to 
be  due  to  a  peculiar  arrangement  of  the  albumen  and  cellulose 
of  the  cell  rather  than  to  any  particular  capsule  around  it. 

Pathogenesis. — When  a  guinea-pig  has  injected  into  its  peri- 
toneal cavity  some  of  the  diluted  sputum  containing  tubercle 
bacilli  it  perishes  in  about  three  weeks,  and  the  following 
picture  presents  itself  at  the  autopsy  :  at  the  point  of  inoculation 
a  local  tuberculosis  shows  itself,  little  tubercular  nodules  contain- 
ing the  characteristic  bacilli.  In  the  lungs  and  the  lymphatics, 
similar  tubercles  are  found,  a  general  tuberculosis. 

If  the  animal  lingers  a  few  weeks  longer,  the  tubercles  become 
necrosed  in  the  centre  and  degeneration  occurs,  the  periphery 
still  containing  active  bacilli,  cavities  having  formed  in  the 
centre. 

Since  the  bacilli  die  in  course  of  time,  killed  by  their  own  pro- 
ducts, their  number  forms  no  correct  guide  of  the  damage  present. 
Even  their  absence  in  the  sputum  does  not  preclude  the  ab- 
sence of  a  tubercular  process.     It  is  their  presence  only  that 


PATHOGENIC    BACTERIA.  105 

warrants  a  positive  declaration.  The  number  of  bacilli  in  a 
given  specimen  is  no  indication  of  the  severity  of  the  disease. 

They  are  found  in  the  blood  only  when  a  vessel  has  come  in 
direct  contact  with  a  tubercular  process  through  rupture  or 
otherwise.  They  have  been  found  in  other  secretions,  milk, 
urine,  etc. 

Man  is  infected  as  follows  : — 

Through  wounds.  —Local  tuberculosis. 

Through  nutrition.— Milk  and  meat  of  tuberculous  animals. 

Phthisical  patients  swallowing  their  own  sputum  and  causing 
an  intestinal  tuberculosis. 

Inhalation. — This  is  the  most  usual  way,  probably  constitu- 
ting the  cause  in  ^  of  the  cases,  except  in  children. 

The  sputum  of  phthisical  patients  expectorated  on  the  floors 
of  dwelling-houses  in  handkerchiefs,  etc,  dries,  and  the  bacilli 
set  free  are  placed  in  motion  by  the  wind  or  rising  with  the  dust 
are  thus  inhaled  by  those  present.  When  the  sputum  is  kept 
from  drying  by  expectoration  in  vessels  containing  water,  this 
great  danger  can  be  avoided. 

Nearly  all  the  cases  of  heredity  can  be  explained  in  this  man- 
ner; the  young  children,  possessing  very  little  resistance,  are 
constantly  exposed  to  the  infection  through  inhalation  and  are 
especially  prone  to  intestinal  infection  through  milk  and  other 
foods. 

Immunity. — No  one  can  be  said  to  be  immune,  though  per- 
sons who  have  been  greatly  weakened  would  offer  less  resistance 
than  healthy  individuals. 

Tuberculosis  in  animals.  Tuberculosis  is  probably  the  most 
widely  disseminated  disease  among  domestic  animals,  and 
affects  cattle,  pigs,  horses,  dogs,  cats,  the  smaller  ruminants, 
birds,  and  even  turtles  and  fish.  The  conclusion  of  Koch, 
made  public  in  his  address  to  the  Tuberculosis  Congress  in 
1901,  that  human  and  bovine  tuberculosis  are  distinct  and  that 
infection  of  human  beings  from  cattle  occurs  so  seldom  that  no 
general  regulations  to  restrict  it  are  necessary,  has  found  few 
adherents.  It  is  true  that  certain  differences  exist  between 
human  and  bovine  tubercle  bacilli,  the  latter  appearing  to  be 
more  virulent  to  animals,  and  it  is  a  fact  that  cattle  are  very 


106        ESSENTIALS  OF  BACTERIOLOGY. 

slightly  susceptible  to  the  human  bacillus,  but  it  is  not  likely 
that  the  converse  is  so.  Children  are  particularly  liable  to  in- 
fection through  the  gastrointestinal  tract,  and  it  has  been 
shown  that  the  uninjured  mucosa  of  the  infant's  intestine  is 
permeable  to  bacillus,  so  that  the  pulmonary  disease  in  the 
young  may  often  be  the  result  of  tuberculous  bronchial  nodes 
secondary  to  tuberculous  glands  of  the  mesentery. 

Various  observations  on  animals  have  shown  that  the  bacillus 
occurring  in  each  species  has  acquired  certain  special  charac- 
teristics regarding  growth  and  virulence.  The  bacilli  causing 
tuberculosis  in  the  cold-blooded  animals  have  departed  farthest 
from  the  human  type,  those  of  birds  to  a  less  degree,  and  those 
of  cattle  least  of  all. 

Products  of  Tubercle  Bacilli.  The  true  nature  of  the  tuber- 
cle toxin  is  not  yet  clear.  It  is  not  unlikely  that  several  toxic 
bodies  differing  from  one  another  in  their  properties  are  pro- 
duced. Koch's  tuberculin  (1890)  was  obtained  by  filtering, 
through  unglazed  porcelain,  concentrated  glycerine  bouillon 
cultures  of  tubercle  bacilli.  It  was  speedily  shown  to  be  devoid 
of  curative  power,  and  is  now  used  mainly  for  diagnosing  the 
disease  in  cattle.  In  healthy  animals  little  or  no  reaction  is 
produced  by  the  injection  of  30  to  40  eg.  of  tuberculin,  but  if 
tubercular,  the  temperature  rises  2°  or  3°  F.  in  eight  to  twelve 
hours,  and  remains  elevated  for  a  like  period  of  time.  In  man 
the  use  of  tuberculin  as  a  diagnostic  measure  is  falling  into  dis- 
favor, as  it  is  both  dangerous  and  unreliable. 

Tuber  culocidin. — This  is  an  albuminoid  obtained  from  the 
original  tuberculin  by  precipitation  with  alcohol.  Klebs  used 
it  as  a  cure  for  tuberculosis. 

Tuberculin  R.  is  an  extract  made  from  dried  and  powdered 
living  bacilli,  and  was  recommended  by  Koch  in  place  of  the 
original  tuberculin,  but  it  has  likewise  proved  useless. 

Agglutination.  Arloing  and  Courmont  have  described  an 
agglutination  reaction  for  the  tubercle  bacillus  similar  to  the 
Widal  reaction  of  typhoid  fever.  It  is  very  unreliable,  how- 
ever, and  but  little  importance  is  attached  to  it. 

Antituberculous  Serum.  The  attempts  to  produce  an  effec- 
tive serum  have  so  far  been  unsuccessful.    Marmorek,  by  grow- 


PATHOGENIC    BACTERIA.  107 

ing  the  bacillus  on  a  special  serum  obtained  by  injecting  calves 
with  the  leucocytes  of  guinea-pigs,  has  secured  a  toxin  which 
he  used  to  immunize  horses,  and  the  serum  so  obtained  has 
been  tried  with  encouraging  results,  but  its  value  is  still  doubt- 
ful. 

Examination  of  Milk  for  Tubercle  Bacilli.  Place  a  drop  of 
the  sample  on  a  cover-glass  and  mix  it  with  2  drops  of  a  1  per 
cent,  solution  of  sodium  carbonate.  The  cover-glass  is  then 
gently  warmed  until  evaporation  is  complete.  The  saponified 
fat  is  then  stained  as  the  ordinary  cover-glass  preparation. 
Only  a  ver}r  few  persons  have  succeeded  in  discovering  the 
bacillus  in  milk. 

Lepra  Bacillus.     (Hansen.) 

Origin. — In  1880  Armauer  Hansen  declared,  as  the  result  ot 
many  years'  investigation,  that  he  found  a  bacillus  in  all  leprous 
processes. 

Form.— Small  slender  rods  somewhat  shorter  than  tubercle 
bacilli,  otherwise  very  similar  in  appearance. 

In  the  interior  of  the  cell  two  to  three  oval  spaces  are  usually 
seen,  not  known  if  spores  or  otherwise. 

Properties.—  They  are  immotile,  do  not  liquefy  the  nutrient 
media. 

Growth.— Bordoni-Uftreduzzi  have  obtained  growths  upon 
blood  serum  to  which  peptone  and  glycerine  had  been  added, 
but  the  accuracy  of  this  observation  is  very  doubtful. 

Staining. — They  resist  the  decolorizing  action  of  acids  as  the 
tubercle  bacilli,  but  they  are  easily  stained,  requiring  but  a 
few  minutes  with  the  ordinary  watery  solutions.  They  take 
Gram's  stain  readily. 

Pathogenesis.— Arning  has  inoculated  prisoners  with  tissue 
obtained  from  leprous  patients,  and  produced  true  leprosy. 

Rabbits  which  had  been  infected  through  the  anterior  chamber 
of  the  eye  showed  the  lepra  nodules  (containing  the  lepra 
bacilli)  diffused  through  various  organs,  but  here  again  the 
results  are  not  wholly  satisfactory. 

In  man  the  skin  and  peripheral  nerves  are  principally  affected, 
but  the  lymphatic  glands,  liver,  and  spleen  can  also  become  the 
seat  of  the  lepra  nodules.     The  lepra  cells  which  compose  these 


108        ESSENTIALS  OF  BACTERIOLOGY. 

nodules  contain  the  bacilli  in  large  numbers.  By  applying  a 
vesicant  to  the  leprous  skin  the  serum  thereby  obtained  will  con- 
tain great  numbers  of  bacilli.     This  is  a  simple  diagnostic  test. 

Method  of  Infection. — Not  yet  determined  ;  the  air,  soil,  water, 
and  food  of  leprous  districts  have  been  carefully  examined  with- 
out result.    The  nasal  secretion  is  very  infectious. 

Syphilis  Bacillus  of  Lustgarten  (Smegma  Bacillus  of  Alvarey 
and  Tavel).  Lustgarten  in  1885,  through  a  certain  staining 
process,  found  peculiar  bacilli  in  syphilitic  tissues  which  he 
thought  had  a  direct  connection  with  the  disease. 

Van  Niessen,  Joseph  and  Piorkowski,  DeLisle  and  Jullien  all 
describe  other  organisms  that  they  have  found  in  syphilitic 
lesions,  and  Schuller  mentions  a  protozoon-like  body  he  has 
seen  in  many  cases,  but  these  results  still  lack  confirmation. 
Metschnikoff,  Roux,  and  Lassar  have  lately  succeeded  in  inocu- 
lating chimpanzees  with  what  appears  to  be  true  syphilis. 

The  question  yet  remains  an  open  one,  what  relation  the 
syphilis  or  the  smegma  bacillus  bears  to  syphilis,  and  will 
remain  so  until  the  bacillus  can  be  cultivated,  which  so  far  has 
not  been  accomplished. 

Bacillus  of  Glanders.  {Bacillus  Mallei,  Loffler-Shutz.)  Botz 
bacillus. 

Origin. — In  the  "  farcy  buds"  or  little  nodules  of  the  disease, 
by  Loffler  and  Shiitz  in  1882. 

Form. — Small  slender  rods,  about  the  size  of  the  tubercle 
bacillus.  The  ends  rounded.  Never  appearing  in  large  collec- 
tions, usually  singly.  Spores  are  said  to  exist,  but  this  is 
doubtful. 

Properties. — The  rods  are  very  resistant,  living  in  a  dried  state 
for  three  months  and  longer  without  any  spores  present.  They 
are  not  motile  ;  possess,  however,  great  molecular  vibration. 

Growth. — The  growth  occurs  between  25°  and  40°  C,  best 
at  37°  C. ;  it  is  very  sparse  upon  gelatine,  but  on  glycerine-agar 
or  blood  serum  a  very  abundant  growth  occurs. 

Colonies. — On  agar  or  glycerine-agar  there  appear  in  two  to 
three  days  small  white  glistening  drops,  which  under  microscope 
seem  as  round  granular  masses  with  an  even  periphery. 

Stroke  Cultures.  —  On  glvcerine-asrar  and  blood  serum  small 


PATHOGENIC    BACTERIA 


109 


transparent   drops    of   whitish   or  grayish   color,   which  soon 
coalesce  to  form  a  broad  band. 

Potato. — An  amber-colored  honey-like  growth  which  gradually 
turns  red,  then  brown,  and  greenish-brown  around  it.  Weakly 
acid  potatoes  are  a  good  medium  and  give  the  most  typical 
growth. 

Fig.  55. 


Bacillus  of  Glanders. 

Staining. — Since  the  bacillus  is  very  easily  decolorized,  some 
special  methods  have  been  recommended. 
Lbffler's.— (For  cover-glass  preparations.) 

1.  Alkaline  methylin  blue  (Loffler's).     5  minutes. 

2.  Acetic  acid  with  a  few  drops  of  tropaeolin.     1  second. 

3.  Washed  in  water. 

For  Sections. — Instead  of  tropseolin  acetic  acid,  the  following 
mixture  is  used  : — 


$.— Oxalic  acid  5  per  cent.        .        .  .     gtt.  j. 

Cone,  sulphuric,  acid gtt.  ij. 

Aq.  destill. 3ij. — M. 

The  sections  are  kept  in  this  5  seconds. 
Kuhne^s  method.     Coverglass. 

1.  Warm  carbol-blue  2  min. 

2.  Decolorized  in  weak  sol.  of  muriatic  acid  (10  parts  to  500). 

3.  Washed  in  water. 


110  ESSENTIALS    OF    BACTERIOLOGY. 

Sections  of  Tissue. 

1.  Carbol-blue,  £  hour. 

2.  Decolorized  in  £  per  cent,  muriatic  acid. 

3.  Washed  in  distilled  water. 

4.  Dehydrated  in  alcohol  1  second. 

5.  Aniline  oil  with  6  gtt.  of  turpentine.     5  min. 

6.  Turpentine,  xylol,  Canada  balsam. 

If  contrast  stain,  add  5  gtt.  of  safranin  (Bismark-brown)  to 
turpentine,  and  use  it  after  the  xylol. 

Pathogenesis. — If  horses,  field  mice,  or  guinea-pigs  be  inocu- 
lated subcutaneously,  with  but  a  very  small  quantity  of  culture, 
a  local  affection  results,  followed  some  time  after  by  a  general 
disturbance  ;  ulcers  form  at  the  point  of  inoculation  ;  little 
nodules,  which  then  caseate,  leaving  scars  and  involving  the 
lymphatics  ;  metastatic  abscesses  then  occur  in  the  spleen  and 
lungs,  and  death  arises  from  exhaustion.  Cattle,  pigs,  and  rab- 
bits are  not  easily  affected ;  man  is  readily  attacked.  The  ba- 
cilli gain  entrance  to  the  blood  and  urine.  Nasal  glanders 
occurs  whatever  the  mode  of  inoculation. 

Manner  of  Infection.— Glanders  being  a  highly  contagious  dis- 
ease, it  requires  but  a  slight  wound  to  allow  it  to  gain  entrance. 

In  horses  the  primary  sore  seems  to  be  at  the  nasal  mucous 
membrane.  In  man  it  is  usually  on  the  fingers.  Boiling  water 
or  1-10,000  sublimate  solution  will  quickly  destroy  the  virulence 
of  this  bacillus. 

Mallein.  A  substance  called  mattein  has  been  obtained  from 
the  cultures  grown  in  glycerin  bouillon.  It  gives  a  reaction 
when  injected  into  cattle  suffering  from  glanders,  and  is  said 
to  be  useful  in  diagnosing  the  disease. 

Bacillus  of  Diphtheria.    (Klebs-Loffler.) 

Origin. — Klebs  found  it  in  membrane  in  1883;  it  was  isolated 
by  Loffler  in  1884. 

Form. — Small,  slightly  curved  rods  about  as  long  as  tubercle 
bacilli  and  twice  as  broad;  the  ends  are  at  times  swollen;  spores 
have  not  been  found.  Their  form  is,  however,  very  variable — 
sometimes  much  longer  than  usual,  one  end  often  greatly 
knobbed.    Normal  bacilli  are  found  only  in  membrane. 

Stained  forms  are  characteristic,  since  the  ends  are  more  easily 


PATHOGENIC    BACTERIA.  Ill 

colored  than  the  centre,  and  usually  the  bacillus  stains  in  seg- 
ments, so  that  it  seems  to  be  made  up  of  very  short  sections. 
At  first  sight  it  appears  like  a  chain  of  cocci. 

Properties.—  They  do  not  possess  any  movement;  do  not 
liquefy  gelatine.  They  are  not  very  resistant,  being  destroyed 
by  a  temperature  of  50°  C,  but  they  have  lived  on  blood-serum 
five  months. 

Growth.— Grow  readily  on  all  media,  but  best  on  blood-serum 
mixtures,  between  temperatures  of  20°  and  40°  C.  They  are  fac- 
ultative anaerobic ;  they  grow  quite  rapidly  and  profusely.  Egg 
cultures  (Hueppe's  method)  give  good  growths.  Passing  cur- 
rents of  air  increase  the  growth. 

Colonies  on  Gelatine  Plates.— At  24°  C.  little  round  colonies, 
white  under  low-power,  granular  centre;  irregular  borders. 

Stab  Cultures.— Small,  white  drops  along  the  needle  track.  In 
glycerine-agar  a  somewhat  profuse  growth. 

Potato.— On  alkaline  surface,  a  grayish  layer  in  48  hours. 

Blood-Serum  (after  Loftier). — Blood  serum  3  parts,  and  bouil- 
lon 1  part ;  the  bouillon  contains  peptone,  1  per  cent.,  chloride 
of  sodium,  £  per  cent.,  and  dextrin  (or  glucose),  1  per  cent. 

In  a  few  hours  (eight  to  sixteen)  on  the  white  opaque  surface 
a  slight  moisture  is  noticeable,  which,  if  examined,  is  composed 
of  bacilli.  In  twenty -four  hours  small  round  colonies  are  found 
which  seem  to  arrange  themselves  concentrically.  The  growth 
becomes  more  abundant,  and  the  individual  colonies  larger  and 
yellowish.  On  blood-coagulum  the  growth  is  usually  gray  and 
the  margins  of  the  culture  crenated.  Often  a  diagnosis  can  be 
made  in  four  hours  if  the  serum  tubes  are  kept  in  a  brood  oven. 

Serum- Agar. — Joos  finds  serum-agar  better  than  Loffler's 
serum  :  300  c.c.  blood-serum  mixed  with  50  c.c.  normal  soda 
solution  and  150  c.c.  water,  heated  in  water  bath  for  2  to  3  hours 
at  60°  to  70°  C,  then  raised  to  100°  O,  or  in  steam  chest  I  hour. 
Then  500  c.c.  peptone  bouillon  (slightly  alkaline)  and  20  gm. 
agar.  When  the  agar  is  dissolved  by  heat,  avoiding  prolonged 
boiling,  the  mixture  is  filtered  and  sterilized  \  hour  at  100°  to 
HO"  C.  in  autoclave ;  then  poured  into  petri  dishes.  Strepto- 
cocci do  not  grow  on  this  medium,  whereas  diphtheria  bacilli 
will  grow  in  from  6  to  12  hours. 


112 


ESSENTIALS  OP  BACTERIOLOGY. 


Bouillon. — In  bouillon  an  abundant  growth  takes  place,  and 
this  medium  is  used  to  obtain  the  toxins. 


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Fig.  50. 


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i 


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V 

ttrM  it*    : 

Bacillus  diphtheria?,  from  a  pure  culture 


Fig.  57. 


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Bacillus  diphtherise,  from  a  culture  upon  blood-serum ;  X  1000  (Fraukel  and  Pfeiffer). 

Staining. — Is  not  colored   by  Gram's  method.     Stained  best 
with  Loffler's  alkaline  methylin-blue. 


PATHOGENIC    BACTERIA.  113 

Pathogenesis. — By  inoculation,  animals,  which  naturally  are 
not  suhject  to  diphtheria,  have  had  diphtheritic  processes  de- 
velop at  the  site  of  infection  ;  hemorrhagic  oedema  then  follows, 
and  death. 

In  rahhits  paralyses  develop,  and  when  the  inoculation  occurs 
upon  the  trachea,  all  the  prominent  symptoms  of  diphtheria 
show  themselves. 

Manner  of  Infection  in  Man. — The  exact  way  is  not  yet  known. 
It  is  supposed  that  the  mucous  membrane  altered  in  some  man- 
ner, the  diphtheria  bacillus,  then  gains  entrance  and  the  disease 
develops.  The  bacilli  may  be  found  in  healthy  individuals  who 
may  act  as  a  source  of  infection  to  susceptible  individuals  with- 
out themselves  becoming  infected. 

Products. — But  it  is  not  the  mere  presence  of  the  bacillus  that 
gives  rise  to  all  trouble  ;  certain  products  which  they  generate 
get  into  the  system  and  produce  the  severe  constitutional  symp- 
toms. 

Roux  and  Yersin,  in  1888,  discovered  that  the  injection  of  the 
filtered  culture  bouillon  (that  is,  freed  of  all  diphtheria  bacilli) 
gave  rise  to  the  same  palsies  as  when  the  bacilli  themselves  were 
introduced. 

Toxins  of  Diphtheria. — Brieger  and  Frankel  filter  the  bouillon 
culture,  evaporate  (in  vacuo  at  27°  C.)  to  i  volume,  then  treat 
with  10  volumes  of  alcohol  and  acetic  acid,  the  precipitate  re- 
dissolved  in  water  and  reprecipitated  with  the  acidulated  alco- 
hol until  a  clear  aqueous  solution  is  obtained ;  this  is  then 
dialyzed  for  72  hours,  and  again  precipitated  with  alcohol,  and 
dried ;  a  white  amorphous  body  results,  giving  all  the  reactions 
of  an  albumen,  and  called  by  them  toxalbumen. 

The  toxin  of  diphtheria,  first  demonstrated  by  Roux  and 
Yersin,  is  not  an  albumen.  It  is  obtained  by  growing  virulent 
bacilli  in  bouillon  for  three  or  four  weeks  at  37°  C.  After  a 
sufficient  alkalinity  has  been  produced  the  cultures  are  fil- 
tered, and  the  filtrate  should  have  a  toxicity  that  will  destroy 
a  500-gramme  guinea-pig  in  twenty-four  hours  when  0.1  c.  cm. 
of  the  toxin  is  injected. 

Antitoxin.     Behring  found  that  animals  rendered  immune 

8 


114        ESSENTIALS  OF  BACTERIOLOGY. 

had  a  principle  in  their  blood  that  was  antagonistic  to  the 
development  of  the  toxin. 

Immunity. — Brieger  and  Frankel,  by  injecting  10  to  20  c.cm. 
of  a  three  weeks'  old  culture  of  diphtheria  bacilli,  which  had 
been  heated  at  70°  C.  for  one  hour,  produced  an  immunity  in 
guinea-pigs  against  the  virulent  form. 

This  important  discovery  has  been  utilized  in  a  practical  Avay. 
Horses  are  made  immune  by  gradually  increased  doses  of  the 
toxin  until  300  c.  cm.  can  be  borne  without  bad  effect.  This 
may  require  several  months'  time.  The  serum  of  such  an  im- 
munized animal  is  now  possessed  of  antitoxic  properties. 

Behring  has  standardized  the  strength  of  antitoxic  serum,  so 
that  we  say  a  serum  has  an  immunizing  strength  of  60  units 
or  100  units,  which  means  that  0.1  c.  cm.  of  the  serum  would 
protect  against  1  c.  cm.  of  the  toxin  when  injected  together  into 
guinea-pigs.  1  cubic  centimetre  of  this  is  the  unit.  The  strength 
commonly  employed  in  human  beings  is  1500  units  in  10  c.  cm. 
If  this  amount  is  injected  into  a  child  suffering  from  diphtheria 
in  the  earlier  stages  (second  to  third  day),  the  disease  is  often 
arrested.  The  membrane  begins  to  disappear,  and  in  two  or 
three  days  has  vanished.  The  constitutional  symptoms  are 
likewise  greatly  influenced  by  the  injection.  If  a  smaller  dose 
is  injected  into  persons  who  have  been  exposed  to  contagion, 
the  disease  is  prevented  from  appearing. 

The  antitoxin  has  no  influence  on  the  bacteria  themselves; 
their  virulence  and  length  of  residence  in  the  body  is  not 
lessened. 

The  toxin  generated  by  the  germ  is  supposed  to  be  neutral- 
ized by  the  antitoxin  and  prevented  from  injuring  the  body 
tissues. 

Pseudo-diphtheria  bacilli,  so  called,  differ  from  the  true 
organism  in  certain  cultural  and  morphological  characteristics, 
and  do  not  produce  a  toxin,  but  their  true  status  is  still  uncer- 
tain. 

Site  of  Bacilli. — Bacilli  are  usually  found  in  the  older  portions 
of  the  pseudo-membrane  very  near  to  the  surface.  The  secre- 
tions of  the  throat  of  a  diphtheritic  child  produced  bacilli  three 
weeks  after  the  temperature  was  down  to  normal. 


PATHOGENIC    BACTERIA. 


115 


Streptococcus  in  Diphtheria.  Streptococci  have  been  found 
quite  constant  in  diphtheria,  but  they  resemble  the  strepto- 
coccus pyogenes,  and  have  no  specific  action. 


Fig.  58. 


Bacillus  typhi,  from  an  agar-agar  culture  six  hours  old,  showing  the  flagella  stained 
by  Loffler's  method  ;  X  1000.(Frankel  and  Pl'eifier.) 

Pseudo-diphtheritic  bacillus  is  probably  a  weakened  or  a  vir- 
ulent form  of  the  true  bacillus. 

Bacillus  of  Typhoid  or  Enteric  Fever.    (Eberth-Gaffky.) 

Origin. — Eberth  found  this  bacillus  in  the  spleen  and  lym- 
phatic glands  in  the  year  1880,  and  Gaff  ky  isolated  and  cultivated 
the  same  four  years  later. 

Form. — Rods  with  rounded  ends  about  three  times  as  Ions  as 
they  are  broad.  Usually  solitary  in  tissue-sections,  but  in  arti- 
ficial cultures  found  in  long  threads.     Flagella  on  the  side. 

Properties. — They  are  very  motile  ;  they  take  the  aniline  dyes 
less  deeply  than  some  similar  bacilli.  Spores  have  not  yet  been 
found ;  they  do  not  liquefy  gelatin. 

Growth. — They  are  facultative  anaerobic;  grow  best  at  37° 
C,  but  can  also  develop  at  ordinary  room  temperature.     All 


116  ESSENTIALS    OF    BACTERIOLOGY. 

nutrient  media  can  be  used  as  culture  ground.  They  develop 
chiefly  on  the  surface,  and  very  slowly.  Repeated  freezing  and 
thawing  do  not  affect  the  vitality  of  the  germ,  and  carbolic  acid 
in  1  to  2  per  cent,  solution  has  no  effect  on  it.  A  ten-minute 
exposure  to  60°  C.  is  invariably  fatal. 

Colonies  on  Gelatine  Plates. — Two  forms  ;  the  ones  near  the 
surface  spread  out  like  a  leaf,  transparent  with  bluish  fluor- 
escence. The  deeper  ones  resemble  whetstone  crystals  of  uric 
acid,  with  the  same  yellowish  tinge. 

In  five  days  they  attain  to  3  millimetres  in  diameter. 

On  Potato  Gelatine. — The  colonies  do  not  have  the  yellow 
color,  they  are  transparent,  later  on  they  become  dark  brown 
with  green  iridescence. 

Stab  Cultures.— Mainly  on  the  surface  a  pearly  layer. 

Stroke  Cultures. — A  transparent  thick  layer. 

Potato. — The  growth  here  is  quite  characteristic.     At  37°  C. 

Fig.  59.  Fig.  60. 


Typhoid  fever  bacillus  in  pure  cul-         Colonies  of  typhoid  bacilli  3  days 
ture.    650  diameters.  old  100  X.    (Frankel  and  Pfeiffer.) 

in  48  hours  a  moist  transparent  film  is  formed  over  the  whole 
surface,  but  so  transparent  that  it  can  hardly  be  seen  without 
close  observation.  If  a  small  portion  of  this  is  placed  under  a 
microscope,  it  will  be  seen  swarming  with  bacilli. 

The  growth  never  becomes  more  prominent ;  the  potato  must 
have  a  neutral  or  acid  reaction. 

Milk. — The  bacteria  grow  very  well  in  milk,  producing  a 
slightly  acid  reaction,  but  no  coagulation. 


PATHOGENIC   BACTERIA.  117 

Carbolized  Gelatine. — Gelatine  which  has  added  to  it  ^  per 
cent,  carbolic  acid  will  allow  the  typhoid  bacillus  to  develop, 
other  similar  bacilli  being  destroyed. 

Glucose  Gelatine. — In  glucose  gelatine  there  is  no  gas-produc- 
tion. Indol  is  likewise  not  generated  by  the  typhoid  bacillus, 
whereas  it  is  by  the  colon  bacillus.  On  Eisner's  potato-gelatine 
the  colon  bacillus  and  the  typhoid  bacillus  grow  readily.  The 
medium  of  Hiss  is  of  great  assistance  in  isolating  the  germ. 

Fig.  61. 


The  Widal  agglutination  reaction  (Slater  and  Spitta). 

The  Gruber- Widal  blood-serum  test,  or,  as  otherwise  known,  the 
agglutination-phenomenon  (Fig.  61),  has  the  following  history: 

About  1889,  Charrin  and  Roger  observed  in  the  serum  of  im- 
munized animals  that  the  B.  pyocyaneus  arranged  itself  in  little 
clumps.  Other  investigators  reported  the  same  thing  for  other 
bacteria,  and  Metschnikoff  added  that  motility  was  destroyed. 

In  1895,  Bordet  showed  that  the  serum  of  cholera-immunized 
animals,  when  mixed  with  bouillon  cultures  of  cholera  spirilla, 
affected  their  motility  and  caused  them  to  form  masses,  or 
"Klumpen,"  as  the  Germans  call  it- 

R.  Pfeiffer,  in  the  same  year,  showed  that  the  introduction  of 
immune  serum  at  the  same  time  with  virulent  cholera  spirilla 


118        ESSENTIALS  OF  BACTERIOLOGY. 

into  the  peritoneum  of  guinea-pigs,  prevented  infection  from 
taking  place,  and  the  spirilla  were  transformed  into  granular 
masses.  He  likewise  showed  this  reaction  to  be  specific,  the 
serum  of  cholera-immune  animals  acting  only  on  cholera 
vibrio;  and  hence  he  suggested  using  the  serum  as  a  means 
of  diagnosis  for  the  cholera  vibrio  and  typhoid  bacillus.  Gru- 
ber  about  the  same  time  made  some  studies  upon  the  use  of 
this  serum  property  in  differentiating  bacteria,  but  it  was  con- 
sidered as  yet  a  property  connected  in  some  way  with  immu- 
nity. 

In  1896,  Widal  and  Griinbaum,  working  separately,  developed 
what  is  now  spoken  of  as  the  " Widal  serum-test,"  or  "Widal 
reaction."  It  consists  in  testing  a  drop  of  blood  of  a  patient 
suspected  of  having  typhoid  fever,  by  mixing  a  dilution  of  it 
with  a  drop  of  a  fresh  bouillon  culture  of  typhoid  bacilli,  and 
examining  the  mixture  in  a  hanging  drop  under  the  micro- 
scope. Within  fifteen  minutes  to  an  hour  the  motility  of  the 
bacilli  will  cease,  and  they  will  have  arranged  themselves  into 
clusters,  as  if  stuck  or  glued  together.  If  this  reaction  occurs 
within  an  hour,  and  with  the  proper  dilution  of  the  serum,  the 
case  is  one  of  typhoid.  Widal  first  used  the  serum  of  the  blood ; 
this  has  been  modified  so  that  even  a  drop  of  dried  blood  is  suf- 
ficient. The  method  as  applied  in  city  laboratories  is  as  follows : 
The  physician  is  told  to  clean  the  finger  of  the  patient  with 
water  (no  germicides),  and  with  a  needle  draw  a  drop  of  blood 
on  to  a  piece  of  ordinary  note-paper.  This  is  then  sent  to  the 
laboratory ;  the  paper  with  the  dried  blood  is  soaked  for  a  few 
minutes  in  a  watch-glass  containing  4  drops  of  clean  water,  thus 
obtaining  a  dilution  of  1 : 5.  One  drop  of  this  is  then  mixed  with 
one  drop  of  a  bouillon  culture  of  typhoid  bacilli  of  about  24 
hours'  growth,  and  examined  under  the  microscope  in  the 
hanging  drop.  Weaker  dilutions  of  the  serum  have  been  rec- 
ommended (1 :  50),  and  this  should  be  used  in  cases  of  doubt 
So  far,  about  95  per  cent,  of  the  cases  examined,  and  which  clin« 
ically  were  considered  typhoid  fever,  have  given  a  positive  reac- 
tion. It  is  not  often  present  until  the  fifth  day  of  the  fever,  and 
disappears  usually  within  a  year,  though  in  some  individuals  it 
has  been  found  ten  years  after  an  attack  of  the  disease. 


PATHOGENIC    BACTERIA.  119 

The  agglutinating  properties  have  been  found  in  nearly  all 
the  secretions  of  the  body — tears,  urine,  milk,  pleuritic  effusions, 
serous  fluid  from  blisters,  etc. 

There  is  no  relation  between  the  reaction  and  the  bactericidal 
power  of  the  serum  ;  the  agglutination  is  not  a  destruction.  The 
agglutinating  power  is  active,  though  the  blood  be  dried  and 
sealed  up  for  months.  It  seems  to  have  no  direct  relation  with 
the  question  of  immunity,  since  it  occurs  at  the  height  of  the 
disease,  and  intense  agglutinating  serum  may  be  had  in  severe 
cases  and  in  cases  with  relapses.  A  negative  result  does  not 
exclude  typhoid. 

The  test  is  quantitative — i.  e.,  it  depends  upon  the  dilution  of 
the  blood-serum,  since  the  serum  of  healthy  persons  in  strong 
dilution  will  cause  agglutination  and  loss  of  mobility. 

The  test  must  occur  within  a  certain  limit  of  time  to  be  of 
value,  since  agglutination  is  liable  to  appear  of  itself  with  non- 
typhoid  sera  after  a  period  of  an  hour. 

As  a  clinical  test  of  the  disease  it  has  considerable  value, 
although  operative  at  a  time  when  other  symptoms  have  devel- 
oped sufficiently  to  determine  the  diagnosis. 

Staining. — Colored  with  the  ordinary  aniline  dyes,  when  they 
are  warmed;  since  they  are  easily  decolorized,  acids  should  be 
avoided. 

Gram's  method  is  not  applicable.  Tissue  sections  stained  as 
follows  : — 

Alkaline  methylin-blue        .  .1  hour. 

Alcohol 5  seconds. 

Aniline  oil 5  minutes. 

Turpentine  oil 1  minute. 

Xylol  and  Canada  bals. 

Such  a  specimen  should  first  be  examined  with  low  power,  to 
focus  little  colored  masses,  then  examined  with  immersion  lens  ; 
these  masses  will  be  found  composed  of  bacilli. 

Similar  Bacteria.  The  Neapolitanus  bacillus  of  Emmerich,  or 
fxces  bacillus  of  Brieger,  resembles  the  typhoid  bacillus  in  many 
ways,  the  colonies  being  the  same  and  its  structure  similar. 
But  the  growth  on  potato  is  very  different;  a  thick,  yellow, 
pasty  layer  is  formed  thereon. 


120  ESSENTIALS    OF    BACTERIOLOGY. 

The  colon  bacillus  not  only  resembles  the  typhoid  germ  in 
form,  but  also  in  some  of  the  pathologic  processes  produced. 
For  points  of  resemblance  and  difference,  see  Bacillus  coli  com- 
munis. 

In  Water.  Bacilli  have  been  found  which  also  resemble  ty- 
phoid bacilli,  and  one  must  be  very  careful  not  to  make  any 
positive  statement. 

Examination  of  Water  for  Typhoid  Bacilli. — When  a  water  is 
supposed  to  contain  typhoid  bacilli,  500  c.cm.  of  the  same  is 
mixed  with  20  gtt.  of  |-per  cent,  carbolic  acid,  which  destroys 
many  of  the  saprophytes. 

Plates  are  then  made  as  described  under  Water  Analysis. 

Those  colonies  which  then  form  and  have  a  tendency  to  liquefy, 
are  touched  on  second  day  with  permanganate  of  potassium, 
and  when  so  colored,  destroyed  with  bichloride  of  mercury. 

Those  that  now  develop  are  transferred  by  inoculation  to  fresh 
plates.  At  the  end  of  eight  days  they  are  examined  under 
microscope  ;  every  colony  not  possessing  motile  bacilli  is  dis- 
carded. The  motile  bacilli  are  tested  with  Gram's  method  of 
staining ;  those  that  do  not  take  the  stain  are  alone  retained. 
Cultures  are  made  from  these  upon  potatoes,  and,  if  the  char- 
acteristic growth  occurs,  then  only  can  they  be  called  typhoid 
bacilli  with  any  certainty. 

Pathogenesis. — Lower  animals  have  npt  yet  been  given  enteric 
fever,  though  their  death  has  been  caused  by  injection  of  the 
bacilli  into  the  veins  of  the  ear. 

In  man  it  has  been  found  in  the  urine,  blood,  sputum,  milk, 
intestinal  discharges,  roseolar  spots,  and  in  various  organs,  as 
spleen,  liver,  lymphatic  glands,  and  intestinal  villi. 

It  is  found  in  secretions  several  days  after  the  attack  has  sub- 
sided.    It  is  found  only  in  this  disease,  and  regularly. 

Way  of  Infection. — The  bacilli  in  the  dejecta  of  the  diseased 
person  find  their  way  into  drinking  water,  milk,  or  dirty  clothes, 
and  so  into  the  alimentary  tract  of  a  person  predisposed  to  the 
disease.  They  enter  the  blood  through  the  lymphatics,  and  so 
become  lodged  in  various  organs.  They  are  quite  resistant,  liv- 
ing for  some  time  in  the  soil  and  water,  and  are  not  affected  as 


PATHOGENIC    BACTERIA.  121 

other  organisms  by  carbolic  acid.  An  epidemic  has  been  traced 
to  the  eating  of  oysters  taken  from  contaminated  water. 

Persistence  in  Water. — Franckland  kept  bacilli  alive  in  water, 
sterilized  by  heat,  75  days ;  in  filtered  water  at  19°  C,  5  days ; 
at  6°  C,  12  days.  In  ordinary  water  they  are  likely  to  be  de- 
stroyed in  a  few  days  by  the  overgrowth  of  other  bacteria. 

Products. — Brieger  found  a  ptomaine  in  the  cultures  which  he 
named  typhotoxin  with  the  formula  C9HnN02.  It  has  no 
specific  action.  A  toxalbumen  insoluble  in  water  has  also  been 
isolated,  but,  as  experiment  animals  are  immune  to  the  disease, 
no  definite  actions  have  yet  been  determined. 

The  cultures, when  old,  show  an  acid  reaction. 

Paracolon  or  paratyphoid  bacilli  are  members  of  the  colon 
group  recently  described  by  Widal,  Gwyn,  Schottmuller,  and 
others.  They  are  of  importance,  since  they  produce  fevers 
clinically  resembling  a  mild  form  of  typhoid,  and  which  are 
rarely  fatal.  They  may  be  the  sole  cause  of  the  disease,  and 
probably  also  occur  together  with  the  typhoid  bacillus  in  mixed 
and  secondary  infections.  Morphologically  they  resemble  the 
typhoid  bacillus,  but  differ  from  it  culturally  and  give  their 
own  serum  reactions  with  the  blood  of  affected  patients.  They 
ferment  glucose,  but  not  lactose  or  saccharose ;  litmus  milk  at 
first  becomes  acid,  but  later  grows  alkaline  and  is  not  coagu- 
lated. On  potato  a  slight  visible  growth  occurs ;  indol  is  usually 
not  formed.  Typhoid  sera  do  not  agglutinate  paracolon  bacilli, 
and  vice  versa;  also  different  paracolon  infections  may  not 
agglutinate  each  other.  The  Bacillus  enteritidis  of  Gartner  is 
a  related  form. 

Bacillus  psittacosis  is  an  allied  form  occurring  in  parrots, 
and  producing  hemorrhagic  septicemia  in  them  and  experi- 
ment animals.  The  disease  is  readily  communicated  to  man 
from  the  affected  birds,  and  causes,  after  ten  days'  incubation, 
a  disease,  the  chief  symptoms  of  which  are  fever,  delirium, 
vomiting,  diarrhoea,  and  albuminuria,  about  a  third  of  the 
cases  ending  fatally.  The  organism  is  agglutinated  by  strong 
dilutions  of  typhoid  serum,  but  the  clumping  is  incomplete  and 
the  bacillus  differs  further  from  the  typhoid  bacillus  in  its 
growth  on  potato  and  in  the  nature  of  the  infection  produced. 


122        ESSENTIALS  OF  BACTERIOLOGY. 

Bacillus  Coli  Communis.    (Escherich.) 

Found  in  human  feces,  intestinal  canal  of  most  animals,  in 
pus  and  water. 

Form. — Short  rods  with  very  slow  movement,  often  associated 
in  little  masses  resembling  the  typhoid  germ,  flagellated,  does 
not  form  spores. 

Fig.  62. 


Bacillus  coli  communis,  from  an  agar-agar  culture;  X  1°00  (Itzerott  and  Niemann). 

Properties.—  Does  not  liquefy  gelatine,  causes  fermentation  in 
saccharine  solutions  in  the  absence  of  oxygen,  produces  acid 
fermentation  in  milk. 

Growth.— On  potato  a  thick,  moist,  yellow-colored  growth. 
Very  soon  after  inoculation  on  gelatine  a  growth  similar  to 
typhoid.  It  can  also  develop  in  carbolized  gelatine,  and  with- 
stands a  temperature  of  45°  C.  without  its  growth  being  de- 
stroyed. 

Pathogenesis.— Inoculated  into  rabbits  or  guinea-pigs,  death 
follows  in  from  one  to  three  days,  the  symptoms  being  those  of 
diarrhoea  and  coma ;  after  death  tumefactions  of  Peyer's  patches 
and  other  parts  of  the  intestine ;  perforations  into  peritoneal 
cavity,  the  blood  containing  a  large  number  of  germs. 

With   the   blood   of  immunized   animals   a  serum   reaction 


PATHOGENIC    BACTERIA. 


123 


similar  to  that  of  typhoid  fever  may  be  obtained  with  cultures 
of  colon  bacilli.  The  colon  bacillus  is  held  responsible  for  most 
of  the  complications  of  typhoid  fever,  such  as  peritonitis, 
cholangitis,  etc.,  by  many  writers. 

Staining. — Ordinary  stains;  do  not  take  Gram. 

Site.—  The  bacillus  has  been  found  very  constant  in  acute 
peritonitis  and  in  cholera  nostras.  Its  presence  in  water  would 
indicate  fecal  contamination,  as  it  is  normally  present  in  the 
intestine. 

Points  of  Resemblance  between  Bacillus  Typhi  and  Bacillus  Coli 
Communis. — 1.  Microscopic  appearance;  2.  Agar  and  gelatine 
cultures ;  3.  Sometimes  growth  on  potato  the  same ;  4.  Stain- 
ing peculiarities ;  5.  Resistance  to  carbolic  acid. 

Points  of  Difference  : 


Colon  Bacillus. 
Less  motile, 
Gelatine  colonies  develop  more 

rapidly, 
Produces  gas   on  dextrose   or 

lactose  media, 
Coagulates  milk, 
Produces  indol, 
Growth  on  potato  visible, 
Changes  neutral  red  to  yellow. 


Typhoid  Bacillus. 
Actively  motile, 
Develop  more  slowly, 

Does  not, 

Does  not, 
Does  not, 
Invisible, 
Does  not  reduce  neutral  red. 


Differences  are  also  noted  in  the  growth  on  special  media, 
such  as  those  of  Hiss  and  Eisner. 

Varieties. — By  some  bacteriologists  the  following  bacilli  are  all 
considered  forms  of  the  colon  bacillus :  B.  lactis  aerogenes  of 
Escherich,  B.  cavicida  of  Brieger,  B.  neapolitanus  of  Emmerich, 
B.  enteritidis  of  Gartner,  and,  together  with  some  other  allied 
organisms,  they  are  spoken  of  as  the  "  colon  group." 


124  ESSENTIALS    OF    BACTERIOLOGY 


CHAPTER  III. 

PATHOGENIC   BACTERIA — CONTINUED. 

Spirillum  Cholerse.     (Koch.)     Comma  bacillus  of  cholera. 

Origin. — Koch,  as  a  member  of  the  German  expedition  sent 

to  India,  in  1883,  to  study  cholera,  found  this  micro-organism 

in   the  intestinal  contents  of  cholera 

Fig.  63.  patients,  and   by  further  experiments 

identified  it  with  the  disease. 

Form.— The  microbe  as  seen  ordi- 
narily appears  as  a  short,  arc-like  body, 
about  half  the  size  of  a  tubercle  bacillus, 
but  when  seen  in  large  groups,  spirals 
are  formed,  each  little  arc  appearing 
then  as  but  a  segment,  a  vibrio ;  each 
arc  is  about  three  times  as  long  as  it 
Comma  bacillus,  pure  cul-     is  broad,  and  possesses  a  flagellum  at 

ture.    600  diameters.  one  or  more  rarely  both  ends. 

Properties. — They  are  very  motile ;  liquefy  gelatine.  They  are 
easily  affected  by  heat  and  dryness.  Spores  have  not  been 
found,  though  some  (Hiippe)  claim  arthrospores,  but  these 
bodies  represent  only  degenerative  changes. 

Growth. — Develops  at  ordinary  temperatures  on  all  nutrient 
media  that  have  an  alkaline  or  neutral  reaction.  They  are 
facultative  anaerobic. 

Colonies,  gelatine. — After  24  hours,  small  white  points  which 
gradually  come  to  the  surface,  the  gelatine  being  slowly  lique- 
fied, a  funnel-shaped  cavity  formed  holding  the  colony  in  its 
narrow  part,  at  the  bottom,  and  on  the  fifth  day  all  the  gelatine 
is  liquid.  If  the  colonies  of  three  days'  growth  are  placed  under 
microscope  they  appear  as  if  composed  of  small  bits  of  frosted 
glass  with  sharp  irregular  points. 


9W 


PATHOGENIC    BACTERIA 


125 


Stab  Culture.— After  30  hours  a  growth  can  be  distinguished 
along  the  needle  track,  and  on  the  surface  a  little  cavity  has 
been  formed,  filled  up  by  a  bubble  of  air,  and  this  liquefaction 
proceeds  until  on  the  sixth  day  it  has  reached  the  sides  of  the 
tube,  tapering,  funnel-shaped  to  the  bottom  of  the  tube.  After 
several  weeks  the  spirilla 

are  found  in  little  collec-  FlGJ-  64- 

tions  at  the  bottom  of  the 
fluid  gelatine.  In  eight 
weeks  the  bacilli  have 
perished. 

Agar. — Stroke  cultures. 
A  shiny  white  layer  lasts 
many  months. 

Potato. — A  yellow  honey- 
like transparent  layer,  if 
the  potato  is  kept  at  ani- 
mal heat. 

Bouillon.  —  A  wrinkled 
scum  is  soon  formed  in 
bouillon.  They  live  wel] 
and  grow  in  sterilized  mill* 
and  sterilized  water,  re^ 
maining  virulent  in  the 
latter  for  many  months. 

In  ordinary  water,  the  bacteria  present  are  destructive  to  the 
comma  bacillus,  and  they  die  in  a  few  days. 

Dunham's  Peptone  Solution. — Useful  for  the  development  of 
nitrites  and  the  indol  reaction. 

WidaVs  serum  test,  as  used  in  typhoid,  is  applicable  in  the 
diagnosis  of  cholera,  using  cholera  cultures  in  place  of  the 
typhoid. 

Staining. — They  are  colored  well  with  watery  aniline  solu- 
tions. The  flagella  can  be  well  seen  by  staining  according  to  the 
flagella  stain. 

Pathogenesis. — Experiment  animals  are  not  subject  to  cholera 
Asiatica,  but  by  overcoming  two  obstacles  Koch  has  produced 
choleraic  symptoms  in  guinea-pigs.  Nieati  and  Rietsch  pre- 
vented peristalsis  and  avoided  the  acidity  of  the  stomach  juices 


Cholera  colonies  after  30  hours  100  X .  (Frankel 
and  Pfeiffer.) 


126        ESSENTIALS  OF  BACTERIOLOGY. 

by  direct  injection  into  the  duodenum,  after  tying  the  gall-duct. 
Koch  alkalinizes  the  gastric  juice  with  5  c.cm.  of  5  per  cent, 
sol.  of  sodii  carbonas,  and  then  injecting  2  grams  of  opium  tinc- 
ture for  every  300  grams  of  weight  into  the  peritoneal  cavity 
paralyzes  peristalsis.  The  cholera  culture  then  introduced 
through  a  stomach-tube,  the  animals  die  in  forty-eight  hours, 
presenting  the  same  symptoms  in  the  appearance  of  the  intes- 
tines as  in  cholera  patients,  the  serous  effusion  containing  great 
numbers  of  spirilla. 

Manner  of  Infection  in  Man.—  Usually  through  the  alimen- 
tary tract,  with  the  food  or  drink,  the  intestinal  discharges  of 
cholera  patients  having  found  entrance  into  the  source  of  drink- 
ing water.  Soiled  clothes  to  fingers,  fingers  to  the  mouth,  etc. ; 
torpid  catarrhal  affection  of  the  digestive  tract  predisposing. 
The  microbe  is  not  found  in  the  blood  or  any  organ  other  than 
the  intestines,  the  tissue  of  the  small  intestines.  It  is  also 
found  in  the  vomit  and  the  intestinal  contents. 

Fig.  65. 


Comma  bacillus  in  mucus,  from  a  case  of  Asiatic  cholera. 

Products. — "  Cholera  red."    When  chemically  pure  nitric  or 
sulphuric  acid  is  added  to  nutrient  peptone  cultures  of  the 


PATHOGENIC    BACTERIA.  127 

cholera  bacillus  a  rose-red  color  is  produced.  This  will  not  take 
place  with  other  bacilli  unless  nitrous  acid  is  present.  The  cholera 
bacillus  forms  nitrites  from  the  nitrates  present  in  the  media, 
and  also  indol.  The  mineral  acid  splits  the  nitrites,  setting  free 
nitrous  acid,  which,  with  the  indol,  forms  the  red  reaction. 
This  pigment  has  been  isolated  and  extracted  and  called 
"  cholera  red."  A  ptomaine,  identical  with  cadaverin,  and  sev- 
eral other  alkaloids  have  been  obtained  from  the  cultures.  A 
toxalbumen  and  a  toxicpeptone  have  lately  been  isolated,  but 
no  special  actions  ascribed  to  them. 

Detection  of  Cholera  Organisms  in  Drinking-water. — When  a  few 
bacteria  are  supposed  to  be  present  in  fecal  matter  or  drinking- 
water  it  is  best  to  add  a  large  quantity  of  the  material  (200 
c.  cm.  of  drinking-water)  to  about  10  c.  cm.  of  bouillon,  and 
place  the  mixture  for  twenty-four  hours  in  an  incubator, 
which  will  cause  rapid  reproduction,  and  then  the  organisms 
can  be  readily  discovered. 

HafFkine  has  obtained  a  great  reduction  in  mortality  in 
cholera  regions  by  the  use  of  anti-cholera  vaccines  as  pro- 
tective and  curative  measures. 

Cholera  Immunity  of  Pfeiffer. — Intraperitoneal,  subcutaneous, 
and  intravenous  injections  of  living  or  dead  cholera  bacteria 
cause  a  disease  in  animals  similar  to  the  cold  stage  of  cholera. 
Death  is  the  result  of  toxemia.  If  the  animal  lives,  the  blood 
has  protective  properties  of  a  specific  nature;  it  has  bacteri- 
cidal properties  against  cholera  vibrio,  and  by  the  injection  of 
this  serum  into  non-immune  animals  it  renders  them  immune. 
The  blood-serum  of  convalescents  and  cholera-vaccinated  indi- 
viduals contains  the  same  bactericidal  substances. 

Bacteria  Similar  to  the  Spirillum  of  Cholera. 

Finkler-Prior  Vibrio,  or  Spirillum  Finkleri. 

Origin. — Found  in  the  intestinal  contents  of  a  patient  suffer- 
ing from  cholera  Asiatica  in  1884,  by  Finkler  and  Prior,  who 
thought  it  identical  with  the  spirillum  of  cholera;  it  differs 
from  it,  however,  in  many  ways,  and  has  been  found  in  healthy 
persons. 

Form. — Somewhat  thicker  than  the  cholera  vibrio:  but  forms 
the  long  spirilla  less  often.     Has  flagella. 


128 


ESSENTIALS  OF  BACTERIOLOGY. 


Liquefies  gelatine  in  a  short 


It 


Fig.  67. 


Properties. — It  is  very  motile 
time. 

Growth. — It  grows  quickly  at  ordinary  room  temperature. 
is  facultative  aerobic. 

Colonies  on  Gelatine  Plates. — Round,  finely  granular  colonfes, 
which  in  twenty-four  hours  are  ten  times  as  large  as  the  cholera 
colonies,  and  in  forty-eight  hours  the  whole  plate  is  liquefied, 
it  being  then  impossible  to  distinguish  any  separate  colonies. 
The  microscopic  appearances  in  no  way 
resemble  the  cholera  colony. 

Stab  Cultures. — The  gelatine  is  lique- 
fied from  above  downwards,  like  a  stock- 
ing in  appearance,  and  in  three  days  is 
completely  liquid. 

Potato.  —  At  ordinary  temperature  a 
thick  gray  layer  covering  the  whole  sur- 
face. 

Water. — It  soon  perishes  in  water. 

Staining. — Ordinary  aniline  dyes. 

Pathogenesis. — For  man  it  has  no  spe- 

Fig.  66. 

Spirillum  Finkleri.    700  diameters.    (Fl 


!ugge.) 


Stab  Culture.    (Finkler- 
Prior.) 


cific  action.  If  it  is  injected  into  Guinea  pigs,  prepared  as 
described  under  the  cholera  bacillus,  they  die,  the  intestines 
having  a  foul  odor,  and  the  bacilli  then  found  in  great  numbers. 

Spirillum  Tyrogenum.    (Deneke.) 

Origin.— In  1885  Deneke  found  in  old  cheese  a  spirillum  very 
similar  in  appearance  to  the  cholera  spirillum. 

Form. — The  same  as  the  cholera  vibrio. 


w   I 
CC  6 

D  1 

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D 
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w  1 

O   1 

^  S 

W 

a 


a  o 


tf  s 

D  ,§ 

H 

H  S 

J   2 

— 

D    1 

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O   & 

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E 

PATHOGENIC    BACTERIA.  129 

Properties. — Very  motile,  liquefy  gelatine. 

Growth. — They  grow  quicker  than  the  cholera,  and  slower 
than  the  Finkler;  they  are  also  facultative  aerobic. 

Colonies. — At  first  resemble  cholera  colonies ;  have,  however, 
a  yellow-green  iridescence,  and  are  more  irregular;  also  grow 
more  rapidly. 

Stab  Cultures. — A  thick  line  along  the  needle-track  and  yellow 
colonies  forming  at  the  bottom,  on  the  surface  a  bubble  of  air 
similar  to  the  cholera.    The  gelatine  is  liquid  in  two  weeks. 

Potato. — At  brood-heat  a  thin  yellow  membrane,  but  not 
always  constant.     Staining,  as  cholera  bacillus. 

Pathogenesis. — When  injected  into  animals  prepared  as  for  the 
cholera  bacillus,  a  certain  number  die. 

Vibrio  Metschnikovi.  (Gamaleia.) 

Origin. — In  the  intestines  of  fowls  suffering  from  a  gastro- 
enteritis, common  in  Kussia.  Gamaleia  found  a  spirillum  which 
bears  so  close  a  resemblance  to  the  cholera  bacillus,  both  in  form 
and  growth,  that  it  cannot  be  distinguished  by  these  character- 
istics alone. 

Form. — As  cholera  bacillus. 

Growth. — Two  kinds  are  found  on  the  gelatine  plate— one  that 
is  identical  in  appearance  with  the  cholera  colony,  the  other  more 
liquefying,  resembling  the  Finkler  spirillum.  If  now  a  second 
plate  be  inoculated  from  either  one  of  these  forms,  both  kinds 
again  are  found  grown,  so  that  it  is  not  a  mixture  of  two  bacilli. 

Stab  Culture. — Similar  to  the  cholera  growth,  a  trifle  faster  in 
growing.    Staining,  as  cholera. 

Pathogenesis.— To  differentiate  it  from  cholera,  these  bacilli, 
when  injected  into  animals,  prove  very  fatal,  and  no  especial 
precautions  need  be  taken  to  make  the  animal  susceptible.  In 
the  pigeon,  guinea-pig,  and  chicken  it  produces  a  hemorrhagic 
oedema,  and  a  septicaemia  which  has  been  called  "  Vibrion 
septicaemia."  The  blood  and  organs  contain  the  spirilla  in 
great  numbers. 

Products.—  The  nitrites  are  formed  just  as  in  cholera  bacillus, 
and  the  red  reaction  given  when  mineral  acids  added  to  gelatine 
cultures.     Certain   products  also   which,   when  injected,   give 


130  ESSENTIALS    OP    BACTERIOLOGY. 

immunity.  The  cultures  are  first  heated  for  one  half  hour  at 
100°  C,  which  destroys  the  germs,  and  then  this  sterilized  pro- 
duct injected.     (5  c.cm.  of  a  five  days'  old  sterilized  culture.) 

In  a  couple  of  weeks  1  to  2  c.cm.  of  the  infected  blood  can  be 
injected  without  causing  any  fatal  result. 

A  great  many  more  spirilla  resembling  the  spirillum  of  chol- 
era have  been  isolated  from  drinking-waters  in  the  past  few 
years,  and  some  bacteriologists  are  inclined  to  consider  them 
as  varieties  of  the  true  cholera  spirillum  which  require  only 
certain  conditions  to  make  them  pathogenic.  Among  these, 
besides  those  already  described,  are  Spirillum  Berolinesis,  S. 
Dunbar,  S.  Danubicus,  S.  Wernicke,  S.  BonhofF,  S.  Weibeli,  S.« 
Schuylkiliensis,  S.  Milleri,  S.  Aquatilis.  The  last  two  are  non- 
pathogenic for  experiment  animals. 

Bacteria  of  Pneumonia.  Two  forms  of  bacteria  have  been 
found  in  this  disease,  and  thought  at  different  times  to  be  the 
cause  of  the  same. 

Neither  one  of  them  is  constant  in  pneumonia ;  and  since 
many  other  pathological  processes  have  shown  them  they  can 
hardly  be  set  down  as  the  sole  cause  of  pneumonia. 

Klebs  in  1875  called  attention  to  the  presence  of  bacteria  in 
pneumonia,  and  in  1882  Friedlander  developed  a  bacillus  from 
the  lung  tissue  of  a  pneumonic  person,  which  he  thought  was  a 
coccus,  and  called  it  pneumococcus. 

In  1886  A.  Frankel  and  Weichselbaum  proved  that  this 
microbe  was  not  constant,  in  fact  was  rare. 

A.  Frankel  obtained  in  the  majority  of  cases  of  pneumonia  a 
microbe  that  he  had  described  in  1884  under  the  name  of 
sputum-septicaemia  micrococcus. 

Weichselbaum  called  it  "  Diplococcus  pneumonias,"  and  be- 
lieved it  to  be  the  real  cause  of  pneumonia.  It  has  been  found 
in  many  other  serous  inflammations,  and  also  in  the  mouths  of 
healthy  persons.  It  is  the  generally  accepted -organism  of  the 
disease,  and  can  be  isolated  from  nearly  all  cases  of  acute 
croupous  pneumonia.  It  is  found  in  about  three-quarters  of 
all  cases  of  pneumonia. 

Streptococcus  pyogenes  and  staphylococcus  pyogenes  aureus  have 
been  found  in  some  cases. 


PATHOGENIC    BACTERIA 


131 


Pneumo-bacillus  (Pneumococcus).     (Friedlander.) 
Origin. — In  the  lung  of  a  croupous-pneumonia  person,  by 
Friedlander,  in  1882. 

Fig.  68. 


Bacillus  pneumoniae  of  Friedlander,  from  the  expectoration  of  a  pneumonia  patient ; 
X  1000  (Frankel  and  Pfeitterj. 

Form.— Small,  almost  oval-shaped  rods,  nearly  as  wide  as 
they  are  long  ;  often  in  pairs,  they  were  at  first  believed  to  be 
cocci.  In  bouillon  cultures  the  rod-form  becomes  more  visible. 
In  tissues  each  bacillus  is  surrounded  by  a  faint  capsule  ;  but 
not  around  those  developed  in  artificial  cultures.  Spores  have 
not  been  found. 

Properties.—  They  are  immobile  ;  do  not  liquefy  gelatine.  A 
gas  is  produced  in  gelatine  cultures. 

Growth.— Grows  rapidly  on  all  media  at  ordinary  temperature  ; 
is  facultative  aerobic. 

Colonies.— On  gelatine  plates.  Small  white  round  colonies, 
reaching  the  surface  in  the  course  of  three  or  four  days  ;  appear- 
ing then  as  little  buttons,  with  a  porcelain-like  shimmer,  the 
edges  smooth. 


132 


ESSENTIALS  OF  BACTERIOLOGY. 


Pig.  69. 


Stab  Culture. — A  growth  along  the  needle-track,  but  on  the 
surface  a  button-like  projection,  which  gives  to  the  growth  the 
appearance  of  a  nail  driven  into  the  gelatine, 
its  head  resting  on  the  surface  ;  therefore 
such  cultures  are  called  "Nail  cultures.'''' 
See  Fig.  69.  Old  cultures  are  colored  brown, 
and  contain  bubbles  of  gas. 

Potato.  — A  yellow,  moist  layer  in  a  few 
days  at  brood-heat.     Gas  bubbles  develop. 

Staining.— The  ordinary  aniline  stains. 
The  sections  do  not  take  Gram's  method  ; 
are  therefore  not  suited  for  double  staining. 

Capsule.— Stained  as  follows  :— 

Cover  glasses. 

1.  Acetic  acid,  two  minutes. 

2.  Allow  acetic  acid  to  dry  by  blowing  air 
upon  it  through  a  glass  tube. 

3.  Saturated,  aniline  water.  Gent,  violet, 
ten  seconds. 

4.  Rinse  in  water.  Mount  in  Canada  balsam. 
For  Sections. 

Bacillus  of  Pneumo-  f  cone,  ale.  gent,  violet,  50.0 

nia.    stab    Culture.     1.  Stain  in  warm  ]  aqua,  100.0 

LNaii  culture.)  I  acetic  acid,  10. 

M.  for  24  hours. 

2.  Rinse  in  one  per  cent,  acetic  acid. 

3.  Alcohol  to  dehydrate.     Mount  in  balsam. 

The  capsule  will  be  found  stained  a  light  blue,  the  bacillus  a 
deep  blue.     (See  also  the  capsule  stain  of  Hiss,  p.  35.) 

Pathogenesis. — Animals  are  not  affected  unless  the  culture  is 
injected  intrapleura. 

Pneumobacillus  of  Frankel.   (A.  Frankel  and  Weichselbaum.) 

Synonyms. — Pneumococcus  ;  Diplococcus  of  Pneumonia  ;  Mi- 
crococcus of  sputum  septicaemia  ;  Micrococcus  Pasteuri ;  Diplo- 
coccus lanceolatus. 

Origin. — A.  Frankel  found  it  in  the  sputum  of  pneumonic 


PATHOGENIC    BACTERIA. 


133 


patients,  thinking  it  at  first  to  be  the  micrococcus  of  sputum 
septicemia  ;  later  he  believed  it  to  be  the  cause  of  pneumonia. 

Form. — Oval  cocci  they  were  at  first  called,  but  they  are  now 
known  to  be  rod-shaped,  being  somewhat  longer  than  broad  ; 
varying,  however,  much  in  size  and  shape.  Usually  found  in 
pairs,  sometimes  in  filaments  of  three  and  four  elements.  In 
the  material  from  the  body  a  capsule  surrounds  each  rod.  In 
the  artificial  cultures  this  is  not  found. 

Fig.  70. 


Bacillus  of  pneumonia  in  blood  of  rabbit  1000  X.    (Frankel  and  Pfeifler.) 


Properties. — They  are  without  self-movement;  do  not  liquefy 
gelatine.     There  are  no  spores. 

Growth.— Grow  only  at  high  temperature,  35°  C.  ;  are  facul- 
tative anaerobic.  The  culture  media  must  be  slightly  alkaline  ; 
the  growth  is  slow. 

Colonies  on  Gelatine  Plates. — Since  the  temperature  must  be 
somewhat  elevated,  the  gelatine  media  need  to  be  thicker  than 
usual  (15  per  cent,  gelatine),  in  order  to  keep  it  solid,  and  a 
temperature  of  24°  C.  used.  Little  round  white  colonies,  some- 
what granular  in  the  centre,  growing  very  slowly. 

Stab  Cultures. — Along  the  needle-track  small  separate  white 
granules,  one  above  the  other,  like  a  string  of  beads. 

Stroke  Culture.  —  On  agar,  transparent,  almost  invisible  little 
drops  resembling  dew  moisture. 


134        ESSENTIALS  OF  BACTERIOLOGY. 

Bouillon. — They  grow  better  here  than  in  the  other  media, 
remaining  alive  a  longer  period  of  time. 

Staining.—  Takes  Gram's  method  and  the  other  aniline  stains 
very  readily.  The  capsule  stained  the  same  way  as  that  of  the 
Friedlander  bacillus. 

Pathogenesis. — Rabbits  and  guinea-pigs,  if  subcutaneously  in- 
jected, die  in  the  course  of  a  couple  of  days  with  septicaemia. 
(0.1  com.  of  a  fresh  bouillon  culture  suffices.) 

Autopsy  shows  greatly  enlarged  spleen  and  myriads  of  bacilli 
in  the  blood  and  viscera,  the  lungs  not  especially  affected.  If 
injected  per  trachea,  a  pneumonia  occurs.  In  man  in  90  per 
cent,  of  croupous  pneumonia  they  are  found  and  usually  only 
during  the  existence  of  the  rusty  sputum,  i.  e.,  the  first  stage. 

Fig.  71. 


1& 

** 


Micrococcus  tetragenus  in  sputum  (tubercle  bacillus  also). 

They  have  also  been  found  in  pleuritis,  peritonitis,  pericarditis, 
meningitis,  and  endocarditis.  They  stand  in  some  intimate  re- 
lation with  all  infectious  inflammations  of  the  body.  Their 
presence  in  healthy  mouth  secretion  does  not  speak  against 
this,  it  requiring  some  slight  injury  to  allow  this  ever-present 
germ  to  develop  into  disease. 

Anti-toxin  of  Pneumonia.    (Klemperer.) 

The  injection  of  very  diluted  cultures  of  the  virulent  bacilli  in- 
travenously has  produced  an  immunity  in  rabbits  and  guinea- 
pigs.  The  serum  of  such  artificially  immune  animals  when  filtered 


PATHOGENIC    BACTERIA.  135 

through  a  Chamberland  filter  and  injected  into  a  rabbit  suffer- 
ing with  pneumonia,  cured  the  same;  or  when  injected  into  a 
susceptible  animal  produced  in  it  immunity  very  quickly.  This 
principle  is  ascribed  to  an  anti-toxin  formed  in  the  tissues  by 
the  diluted  proteids,  and  this  anti-toxin  neutralizes  the  toxicity 
of  the  strong  virus. 

Bacillus  of  Rhinoscleroma.  (Frisch.  1882.)  It  was  found  in 
the  tissue  of  a  rhinoscleroma,  but  resembles  the  Friedlander 
bacillus  in  nearly  every  respect,  and  as  the  disease  rhinoscleroma 
was  not  reproduced  by  the  inoculation  of  the  bacillus  in  animals, 
it  can  be  considered  identical.  The  growth,  cultures,  and  pro- 
perties are  the  same  as  the  pneumobacillus  of  Friedlander. 

Diplococcus  Intracellularis  Meningitidis.  Weichselbaum 
claims  to  have  found  a  special  diplococcus  in  epidemic  cerebro- 
spinal meningitis,  which  differs  in  a  few  respects  from  the  pneu- 
mococcus  of  Frankel :  growth  most  abundant  on  blood-serum — 
round,  white,  shining  colonies  in  twenty-four  hours.  It  does 
not  take  Gram's  stain ;  does  not  affect  animals  when  injected 
eubcutaneously.  Inoculated  into  the  meninges  of  the  dog  and 
goat,  a  meningitis  has  been  produced,  and  when  found  in  the 
exudate  of  the  meninges  lies  in  the  protoplasm  and  nuclei  of 
the  leucocytes.  The  organism  has  many  points  in  common 
with  the  gonococcus,  but  differs  from  it  in  the  ease  of  cultivation. 

Micrococcus  Tetragenus.     (Koch.    Gaffky). 

Origin. — Koch  found  this  microbe  in  the  cavity  of  a  tuber- 
culous lung.  Gaffky,  in  1883,  studied  its  pathogenic  actions 
and  gave  it  the  name  it  now  bears. 

Form. — Cocci  which  are  gathered  in  the  tissues  in  groups  of 
four,  forming  a  square,  a  tetrad.  See  Fig.  71.  In  artificial 
culture,  sometimes  found  in  pairs.  A  capsule  of  light  gelat- 
inous consistence  surrounds  each  tetrad. 

Properties. — They  are  immobile ;  do  not  liquefy  gelatine. 

Growth. — They  grow  well  on  all  nutrient  media  at  ordinary  and 
brood  temperatures ;  are  facultative  aerobic.    They  grow  slowly. 

Colonies  in  gelatine  plates.  In  two  days,  little  white  spots, 
which  when  on  the  surface  form  little  elevations  of  a  porce- 
lain-like appearance;  under  low  power  they  are  seen  very 
finely  granulated. 


136 


ESSENTIALS  OF  BACTERIOLOGY. 


Fig.  72. 


v\ 


Stab    Culture. — Small    round    separated    colonies    along  the 
needle-track,  and   on   the  surface   a  button-like   elevation,  a 
form  of  "  nail  culture."     See  Fig.  72. 

Potato. — A  thick  slimy  layer  which  can  be 
loosened  in  long  shreds. 

Staining.—  Colored  with  the  ordinary  ani- 
line stains.     Gram's  method  also  applicable. 
Pathogenesis.— White  mice  and  guinea-pigs 
die  in  a  few  days  of  septicaemia  when  injected 
with  the  tetragenus  cultures,  and  the  micro- 
coccus is  then  found  in  large  numbers  in  the 
blood  and  viscera.     Field  mice  are  immune. 
In  the  cavities  of  tubercular  lungs,  in  the 
sputum  of  phthisical  and  healthy  patients,  it 
is  often  found,  but  what  action  it  has  upon 
man  has  not  yet  been  determined. 
Capsule  Bacillus.    (Pfeiffer.) 
Origin. — Stringy   exudate  and  blood   of  a 
dead  guinea-pig. 

Form. — Thick  little  rods,  sometimes  in  long 
threads.  Large  oval  capsules  in  the  stained 
preparations. 

Properties. — Immotile,  not  liquefying,  an 
odorless  gas  in  gelatine  cultures. 

Growth. — At  ordinary  temperatures,  rap- 
idly; facultative  anaerobin. 

Gelatine  Plates. — Oval  points,  and  like  a  por- 
celain button  on  the  surface. 

Stab  Cultures. — Like  the  pneumonia  bacillus 
of  Friedlander. 

Potatoes. — Abundant  growth,  yellow  color 
and  moist,  coming  off  in  strings. 
Staining. — Hot  fuchsin  colors  the  capsule  intensely ;  carefully 
decolorizing  with  acetic  acid,  the  capsules  are  red  or  light  violet 
around  the  deeply-tinged  bacillus.     Gram's  method  not  applic- 
able. 

Pathogenesis. — Subcutaneously  injected  in  mice,  they  die  in 
48  hours.     Rabbits  die  when  a  large  quantity  is  injected  into 


Stab  Culture. 
Micrococcus  tetra- 
genus. 


PATHOGENIC    BACTERIA.  137 

the  circulation.  The  blood  and  juices  have  a  peculiar  stringy 
fibrinous  consistence. 

Bacillus  of  Influenza.    (Pfeiffer,  1892.) 

A  small  bacillus  about  one-half  the  size  of  the  bacillus  of 
mouse  septicaemia,  and  arranged  in  chain-form.     It  develops 

Fig.  73. 


»     .  •  *  <« 


Bacillus  influenzae,  from  a  gelatin  culture;  X  1000  (Itzerott  and  Niemann). 

upon  blood-serum  agar.  It  is  aerobic.  Without  movement; 
does  not  take  the  Gram  stain.  It  is  best  stained  with  diluted 
carbol-fuchsin,  the  contrast-stain  being  Loffler's  methylene- 
blue.  Upon  glycerine-agar,  over  which  a  drop  of  blood  has  been 
spread,  in  an  incubator  at  the  end  of  twenty-four  hours  a  very 
delicate  growth  occurs,  which  resembles  condensed  moisture. 
It  is  found  in  the  sputum  and  in  the  bronchial  nasal  secretions 
and  blood  of  influenza  patients,  but  cannot  as  yet  be  said  to  be 
the  cause  of  influenza. 

Micro-Organisms  of  Suppuration.  The  suppuration  of  wounds 
is  due  to  the  presence  of  germs.  The  knowledge  of  this  fact  is 
the  basis  of  the  antiseptic  treatment  in  surgery  ;  for  when  the 
microbes  can  be  destroyed  or  their  entrance  prevented,  the 
wounds  are  made  clean  and  kept  without  suppurating.  Vari- 
ous forms  of  bacteria  have  been  found  in  septic  processes,  and 


138 


ESSENTIALS  OF  BACTERIOLOGY-. 


the  formation  of  pus  cannot  be  ascribed  to  any  particular  one 
alone ;  some,  more  common  than  others,  are  found  in  nearly  all 
forms  of  suppuration ;  others  give  rise  to  special  types. 

Wounds  are  often  irritated  by  for- 
FlG#  74,  eign  bodies  and  chemicals,  and  a  dis- 

charge occurs  in  them  even  when 
every  aseptic  and  antiseptic  precau- 
tion has  been  taken ;  but  such  a  dis- 
charge is  free  from  bacteria,  and  no 
more  like  pus  than  a  benign  growth 
is  like  a  malignant  one. 

Fig.  75. 


Streptococcus  pyogenes:  cult- 
ure upon  agar-agar  two  days  old 
(Frankel  and  Pfeiffer). 


Streptococcus  pyogenes  (Jakob). 


Streptococcus  Pyogenes.  (Rosenbach.)  Streptococcus  erysipe- 
latis.     (Fehleisen.) 

Origin. — Fehleisen  discovered  this  microbe  in  the  lymphatics 
of  the  skin  in  erysipelas,  and  he  thought  it  the  cause  of  the 
same.  Under  the  name  streptococcus  pyogenes,  Rosenbach 
described  an  identical  coccus  which  has  been  found  in  nearly 
all  suppurative  conditions. 

Form. — Small  cocci  singly  and  in  chain-like  groups.     Spores 


PATHOGENIC    BACTERIA.  139 

have  not  been  found,  though  it  is  supposed  because  of  their 
permanency  that  spores  are  present. 

Properties. — They  are  immotile,  do  not  liquefy  gelatine. 

Growth. — They  grow  slowly,  usually  on  the  surface,  and  best 
at  higher  temperatures. 

Colonies. — In  three  days  a  very  small  grayish  speck,  which 
hardly  ever  becomes  much  larger  than  a  pin-head  ;  under  micro- 
scope, looking  yellowish,  finely  granular,  the  edges  quite  defined. 

Stub  Cultures. — Along  the  needle-track  little  separated  colonies 
like  strings  of  beads,  which  after  a  time  become  one  solid  white 
string. 

Stroke  Culture. — Little  drops,  never  coalescing,  having  a  bluish 
tint. 

Potato. — No  apparent  growth. 

Bouillon.—  At  37°  C.  clouds  are  formed  in  the  bouillon,  which 
then  sink  to  the  bottom,  and  long  chains  of  cocci  found  in  this 
growth. 

Staining. — Easily  colored  with  the  ordinary  stains.  Gram's 
method  is  also  applicable. 

Pathogenesis. — Inoculated  subcutaneously  in  the  ear  of  a 
rabbit,  an  erysipelatous  condition  develops  in  a  few  days, 
rapidly  spreading  from  point  of  infection. 

In  man,  inoculations  have  been  made  to  produce  an  effect 
upon  carcinomatous  growths.  Erysipelas  was  always  produced 
thereby.  When  it  occurs  upon  the  valves  of  the  heart,  endo- 
carditis results.  Puerperal  fever  is  caused  by  the  microbe  in- 
fecting the  endometrium,  the  Streptococcus  puerperalis  of  Frankel 
being  the  same  germ. 

In  scarlatina,  variola,  yellow  fever,  cerebro-spinal  meningitis, 
and  many  similar  diseases,  the  microbe  has  been  an  almost  con- 
stant attendant.  It  is  often  associated  with  the  diphtheria 
bacillus  in  true  diphtheria,  and  is  the  cause  of  many  of  the 
diphtheritic  affections  of  the  throat  in  which  the  diphtheria 
bacillus  is  absent. 

An  antistreptococcic  serum  has  been  used  as  a  curative 
agent  in  puerperal  fever,  scarlatina,  and  other  diseases  sup- 
posed to  be  due  to  this  germ. 

A  mixture  of  a  culture  of  Pyogenes  and  Prodigiosus  has  been 


140        ESSENTIALS  OF  BACTERIOLOGY. 

used  as  an  injection,  with  apparent  benefit,  in  inoperable  cases 
of  sarcoma. 
Staphylococcus  Pyogenes  Aureus.    (Rosenbach.) 

Fig.  76. 


Staphylococcus  pyogenes  albus  (Jakob). 

Origin. — Found  commonly  in  pus  (80  per  cent,  of  all  suppura- 
tions), in  air,  water,  and  earth ;  also  in  sputum  of  healthy  persons. 

Form. — Micrococci  in  clusters  like  bunched  grapes,  hence  the 
name  staphylo,  which  means  grape.  They  never  form  chains. 
Spores  have  not  been  found,  though  the  cocci  are  very  resistant. 

Properties. — Without  movement ;  liquefying  gelatine.  It  gives 
rise  to  an  orange-yellow  pigment  in  the  various  cultures. 

Growth. — It  grows  moderately  fast  at  ordinary  temperature, 
and  can  live  without  air,  a  facultative  serobin  and  anterobin. 

Colonies  on  Gelatine. — On  second  day  small  dots  on  the  surface, 
containing  in  their  centre  an  orange-yellow  spot.  The  gelatine 
all  around  the  colony  is  liquefied  ;  the  size  is  never  much  greater 
than  that  attained  the  second  day. 

Colonies  on  Agar. — The  pigment  remains  a  long  time. 

Stab  Culture. — At  first,  gray  growth  along  the  track,  which, 
after  three  days,  has  settled  at  the  bottom  of  the  tube  in  a  yel- 
low granular  mass,  the  gelatine  being  all  liquid. 

Stroke  Culture  on  Agar. — The  pigment  diffused  over  the  sur- 
face where  the  growth  is,  in  moist  masses. 


PATHOGENIC    BACTERIA. 


141 


Fig. 


Potato. — A  thin  white  layer  which  gradually  becomes  yellow 
and  gives  out  a  doughy  smell. 

Staining.— Very  readily  colored  with  ordinary  stains;  also 
with  Gram's  method. 

Pathogenesis.— When  rabbits  are  injected  with  cultures  of  this 
microbe  into  the  knee-joint  or  pleura,  they  die  in  a  day.  If 
injected  subcutaneously,  only  a  local  action  occurs,  namely, 
abscesses. 

If  directly  into  circulation,  a  general  phleg- 
monous condition  arises,  the  capillaries  be- 
come plugged  with  masses  of  cocci,  infarct 
occur  in  kidney  and  liver,  and  metastatic  ab- 
scesses form  in  viscera  and  joints.  Garre\  by 
rubbing  the  culture  on  his  forearm,  caused 
carbuncles  to  appear. 

Several  varieties  of  the  pyogenic  staphylo- 
cocci are  recognized  according  to  their  color- 
producing  properties  and  slight  variations  of 
growth.  Of  these,  the  staphylococcus  pyog- 
enes aureus  is  the  most  virulent,  and  is  con- 
sidered the  type  of  the  group.  They  are 
always  present  on  the  surface  of  the  body, 
beneath  the  nails,  in  the  nose  and  mouth,  in 
the  dust  of  streets,  and  on  the  floor  of  houses. 

Staphylococcus    pyogenes    albus    differs 
from  the  preceding  only  in  the  absence  of  Stab  culture.  Micro- 
pigment  and  in  its  slight  virulence.     Welch     coccus  Py°genes 

°  aureus. 

describes  a  variety  constantly  found  both  on 
the  skin  and  in  its  deeper  layers,  which  he  calls  the  staphylo- 
coccus epidermidis  albus. 

Micrococcus  Pyogenes  Citreus.  (Passett.)  This  liquefies  gel- 
atine less  rapidly  than  the  pyogenes  aureus,  and  forms  a  citron- 
yellow  pigment  instead  of  the  orange-yellow  of  the  aureus. 

Micrococcus  Cereus  Albus.  (Passet.)  Differs  from  the  pyo- 
genes albus  in  the  form  of  colony.  A  white  shiny  growth  like 
drops  of  wax;  hence  the  name  cereus. 

Micrococcus  Cereus  Flavus.  (Passet.)  A  lemon-yellow 
colored  growth  after  a  short  time,  otherwise  not  differing  from 
cereus  albus. 


142  ESSENTIALS   OF   BACTERIOLOGY. 

Micrococcus  Pyogenes  Tenuis.    (Rosenbach.) 

Origin.— Found  in  the  pus  of  large  inclosed  abscesses. 

Form. — Cocci,  without  any  especial  arrangement. 

Properties. — Not  much  studied. 

Growth. — Cultivated  on  agar,  it  forms  clear  thin  colonies;  along 
the  needle-track  an  opaque  streak,  looking  as  if  varnished  over. 

Bacillus  Pyocyaneus.    (Gessard.) 

Synonyms. — Bacterium  seruginosum,  bacillus  fluorescens. 
(Schroter.)     The  bacillus  of  bluish-green  pus. 

Origin. — Found  in  1882  in  the  green  pus  in  pyocyeemia. 

Form. — Small  slender  rods  with  rounded  ends,  easily  mistaken 
for  cocci.     Often  in  groups  of  four  and  six,  without  spores. 

Properties. — Very  motile  ;  liquefy  gelatine  rapidly  ;  a  peculiar 
sweetish  odor  is  produced  in  the  cultures,  and  a  blue  pigment. 

Fig.  78. 


*«*N*«- 


Bacillus  pyocyaneus,  from  an  agar-agar  culture ;  X  1000  (Itzerott  and  Niemann). 

Growth. — Develops  readily  at  ordinary  temperature,  growing 
quickly  and  mostly  on  the"  surface  ;  it  is  aerobic.  Colonies  on  gela- 
tine plate,  in  two  or  three  days  a  greenish  iridescence  appears 
over  the  whole  plate,  the  colonies  having  a  funnel-shaped  lique- 
faction, and  appearing  under  low  power  when  still  young,  as 
yellowish  green,  the  periphery  being  granulated. 

Stab  Cultures. — Mainly  in  upper  strata,  the  liquefaction  funnel- 
shaped,  the  growth  gradually  settling  at  the  bottom,  a  rich  green 


PATHOGENIC    BACTERIA.  143 

shimmer  forming  on  the  surface,  and  the  gelatine  having  a  deep 
fluorescence. 

Potato. — The  potato  is  soaked  with  the  pigment,  a  deep  fold 
of  green  occurring  on  the  surface. 

Staining. — With  ordinary  aniline  dyes. 

Pathogenesis. — When  animals  are  injected  with  fresh  cultures 
in  the  peritoneal  cavities  or  cellular  tissues,  a  rapidly  spreading 
oedema  with  general  suppuration  develops.  The  bacilli  are 
found  in  the  viscera  and  blood. 

If  a  small  quantity  is  injected,  a  local  suppuration  occurs,  and 
if  the  animal  does  not  die  it  then  can  withstand  large  quanti- 
ties.    It  is  immune. 

The  Pigment.  Pyocyanin. — When  the  pus,  bandages,  and 
dressings  containing  the  bacillus  pyocyaneus  are  washed  in 
chloroform,  the  pigment  is  dissolved  and  crystallizes  from  the 
chloroform  in  long  needles.  It  is  soluble  in  acidulated  water, 
which  is  turned  red  thereby,  and  when  neutralized  the  blue  color 
returns.  It  has  no  pathogenic  action.  It  is  an  aromatic  com- 
pound. The  bacillus  has  no  especial  action  on  the  wound,  and 
is  found  sometimes  in  perspiration  of  healthy  persons. 

Bacillus  Pyocyaneus.  |3.  (Ernst.)  A  bacillus  found  in  gray- 
ish pus-colored  bandages. 

The  only  especial  difference  between  this  and  the  above  is  the 
formation  of  brownish-yellow  pigment  instead  of  pyocyanin.  The 
form  and  appearance  of  cultures  otherwise  the  same. 

Micrococcus  Gonorrhoeae.    Gonococcus.     (Neisser.)    In  1879 
Neisser  demonstrated  the  presence  of  this 
germ  in  the  secretion  of  specific  urethritis.  Fi®.  79. 

Form. — Cocci,  somewhat  triangular  in 
form,  found  nearly  always  in  pairs,  the  base 
of  one  coccus  facing  the  base  of  the  other, 
and  giving  the  appearance  of  a  Vienna  roll, 
hence  the  German  name  Semmel  (roll)-form. 

:■%. 

•v»-  ' 

Immotile. 

Culture. — On  gelatine-agar  or  potato  they 
do  not  grow,  and  only  upon  human-blood         Gonococci  in  gon- 
serum  have  they  given  any  semblance  of  a       nne^methyi'  *toi£ 
growth.     The  temperature  must  be  between       (650  diameters.) 


Four  to  twelve  such  pairs  are  often  found 
together.  r 


144 


ESSENTIALS  OF  BACTERIOLOGY. 


83°    and    37°    C,    and    the    growth    occurs   very   slowly   and 
sparsely. 

Method  of  Cultivation  (Wertheim).— Gonorrheal  pus  is  mixed 
in  a  test-tube  with  liquid  human  blood  serum  of  40°  C.  temper- 
ature, and  two  dilutions  are  made  with  blood  of  the  same  tem- 
perature. An  equal  quantity  of  2  per  cent,  agar  solution  is 
now  poured  into  each  tube,  and  three  glass  dishes  are  covered 
at  once  with  this  mixture.  After  being  in  the  brood  oven  for 
twenty-four  hours  colonies  can  be  discovered. 


Fig.  80. 


Gonococcus in  urethral  pus;  X  1000  (Frankel  and  Pfeiffer). 

In  three  days  a  very  thin,  almost  invisible,  moist  3rellowish 
growth,  seemingly  composed  of  little  drops. 

Under  low  power  small  processes  are  seen  shooting  out  from 
the  smooth  border. 

It  requires  to  be  then  transferred  to  fresh  media,  as  it  quickly 
perishes. 

Cultivation  has  also  occurred  on  acid  gelatine,  gelatine  con- 
taining acid  urine,  in  acid  urine  itself,  and  albuminous  urine 
with  agar. 

Staining. — Colored  easily  with  all  ordinary  aniline  stains. 


PATHOGENIC   BACTERIA.  145 

Gram's  method  is  not  applicable,  this  being  one  of  its  main 
diagnostic  features. 

The  following  method  is  recommended  by  Neisser. 

The  cover-glasses,  with  some  of  the  urethral  discharge  smeared 
upon  them,  are  covered  with  a  few  drops  of  alcoholic  solution  of 
eosin  and  heated  for  a  few  minutes  over  the  flame.  The  excess 
of  the  dye  is  removed  with  filter  paper,  then  the  cover-glass 
placed  in  concentrated  methylin  blue  (alcoholic  solution)  for  15 
seconds,  and  rinsed  in  water. 

The  gonococci  are  dark  blue,  the  protoplasm  of  the  cell  pink, 
and  the  nucleus  a  light  blue,  the  gonococci  lying  in  the  proto- 
plasm next  to  the  nucleus. 

Other  bacteria  are  similar  to  the  gonococci  in  form  ;  they  are 
distinguished  from  the  gonococcus,  in  that  they  are  colored  with 
Gram's  method,  whereas  the  micrococcus  of  gonorrhoea  is  not. 
The  points  on  which  the  diagnosis  is  to  be  made  are  the  char- 
acteristic biscuit  shape,  the  intracellular  position  of  the  organ- 
ism, and  its  failure  to  stain  with  Gram. 

Pathogenesis. — The  attempts  to  infect  the  experiment  ani- 
mals with  gonorrhoea  have  so  far  been  without  success.  In  man, 
upon  a  healthy  urethra,  a  specific  urethritis  was  produced  with 
even  the  20th  generation  of  the  culture.  Gonorrhoeal  ophthalmia 
contains  the  cocci  in  great  numbers,  and  endocarditis  and  gon- 
orrhceal  rheumatism  are  said  to  be  caused  by  the  cocci. 

The  microbes  have  been  found  long  after  the  acute  attack, 
when  only  a  very  slight  oozing  remained,  and  the  same  were 
very  virulent. 

The  specific  inflammations  of  the  generative  organs  of  the 
female  are  due  to  this  microbe,  having  gained  entrance  through 
the  vagina,  extending  its  influence.  It  is  found  chiefly  in  the 
superficial  layers  of  the  mucous  membrane. 

A  temperature  of  40°  C.  for  12  hours  destroys  the  gonococci. 

Gonotoxin.—A  toxin  has  been  isolated  which  causes  fever,  loss 
of  weight,  and  finally  death.  The  urethra  is  not  immunized  by 
repeated  injections.  In  man  the  toxin  causes  painful  indura- 
tions lasting  several  days. 

Similar  Microbes  found  in  the  Urethra  and  Vagina. 

Micrococcus  Citreus  Conglomerata.     (Bumra.)     Similar  to 

10 


116  ESSENTIALS    OP    BACTERIOLOGY. 

the  gonococci  in  form,  they  are,  however,  easily  cultivated,  and 
form  yellow  colonies  which  dissolve  the  gelatine  and  grow  rap- 
idly; the  surface  of  the  gelatine  is  at  first  moist  and  shiny,  but 
later  on  wrinkled.  Colored  with  Gram's  method,  and  have  no  spe- 
cial pathological  action.    Found  in  the  air  and  gonorrhoeal  pus. 

Diplococcus  Albicans  Amplus.  (Bumm. )  In  vaginal  secretion. 
The  diplococci  are  much  larger  than  the  gonococci,  but  similar 
in  form.  They  are  also  cultivated  upon  gelatine  plates,  grayish- 
white  colonies,  which  slowly  liquefy  gelatine.  They  grow  mode- 
rately rapid.  Stained  with  Gram's  method,  and  have  no 
pathogenic  action. 

Diplococcus  Albicans  Tardissimus.    (Bumm.) 

Origin. — In  urethral  pus.  Form,  like  gonococci.  Properties, 
immotile ;  do  not  liquefy  gelatine.  Growth,  very  slow  at  ordi- 
nary temperature,  but  more  rapid  at  brood-heat.  The  colonies 
are  small  white  points,  which  under  low  power  appear  brown 
and  opaque. 

Agar  Stroke  Culture. — Grayish-white  growth,  which  after  two 
months  is  like  a  skin  upon  the  surface. 

Staining. — Takes  Gram's  method. 

Pathogenesis. — None  known. 

Micrococcus  Subflavus.    (Bumm.) 

Origin.— In  lochial  discharges,  in  vagina  and  urethra  of 
healthy  persons. 

Form. — As  gonococci. 

Properties. — Not  motile  ;  liquefy  gelatine  slowly  ;  a  yellow- 
brownish  pigment. 

Growth.  —  Grows  slowly  on  all  media,  forming  on  gelatine, 
after  two  weeks,  a  moist  yellowish  surface  growth. 

Potato.  —  Small  half-moon-shaped  colonies  which,  after  three 
weeks,  become  light-brown  in  color,  and  covering  the  surface  as 
a  skin. 

Staining. — Colored  ivith  Gram. 

Pathogenesis. — Not  acting  upon  the  mucous  membrane,  but 
when  injected  in  cellular  connective  tissue,  an  abscess  results 
which  contains  myriads  of  diplococci. 

The  gonococcus  is  distinguished  from  all  these  similar  micro- 
cocci by  being  found  usually  within  the  cell  protoplasm. 


PATHOGENIC    BACTERIA.  147 

Secondly.— Not  stained  with  Gram's  method. 

Thirdly. — Refusing  to  grow  readily  upon  gelatine. 

All  the  similar  bacteria  being  easily  cultivated. 

These  characteristics,  taken  in  toto,  form  sufficient  features  for 
its  ready  recognition,  and  as  it  is  often  a  serious  question  to 
decide,  not  so  much  because  of  the  patient's  health  as  because 
of  his  character,  we  should  be  very  careful  not  to  pronounce  a 
verdict  until  we  have  tested  the  micro-organism  as  above.  When 
the  germ  so  tested  is  found,  the  process  can  be  called  specific 
without  a  doubt. 

Fig.  81. 


/ 


*  :^v 


Bacillus  of  Tetanus  with  spores. 

Bacillus  of  Tetanus.    (Nicolaier-Kitasato.) 

Oi*igin.—  Nicolaier  found  this  bacillus  in  the  pus  of  a  wound 
in  one  who  had  died  of  tetanus,  describing  it  in  1884. 

Kitasato  has  since  then  been  able  to  isolate  and  cultivate  this 
germ.  (1889.) 

Form.— A  very  delicate,  slender  rod,  somewhat  longer  than 
the  bacillus  of  mouse  septicaemia,  which  is  the  smallest  bacillus. 

When  the  spores  form,  a  small  swelling  occurs  at  the  end 
where  the  spore  lies,  giving  it  a  drum-stick  shape. 


148 


ESSENTIALS  OF  BACTERIOLOGY. 


Properties.-  JSot  very  motile,  though  distinctly  so ;  liquefies 
gelatine  slowly.     The  cultures  give  rise  to  a  foul-smelling  gas. 
Growth.— Develops  very  slowly,  best  at  brood-heat  (36°  to  38° 


Fig.  82. 


Fig.  83. 


tf 


Racillus  tetani :  culture  four  days 
old  in  glucose-gelatine  (Frankel  and 
Pfeiffer). 


Six  days'  culture  of  bacillus 
of  tetanus  in  gelatine  (deep 
stab).     (Frankel  and  Pfeiffer.) 


C),  and  only  when  all  oxygen  is  excluded,  an  obligatory  ancero- 
bin.  In  an  atmosphere  of  carbon  dioxide  gas  it  cannot  grow, 
but  in  hydrogen  it  nourishes. 


PATHOGENIC    BACTERIA.  149 

Colonies  on  gelatine  plates  in  an  atmosphere  of  hydrogen. 
Small  colonies.  After  four  days  a  thick  centre  and  radiating 
wreath-like  periphery,  like  the  colonies  of  bacillus  subtilis. 

Hujh  Stab-Culture. — (The  gelatine  having  2  per  cent,  glucose 
added  and  filling  the  tube.)  Along  the  lower  portion  of  the  needle- 
track,  a  thorny-like  growth,  little  needle-like  points  shooting 
out  from  a  straight  line.  The  whole  tube  becomes  clouded  as 
the  gelatine  liquefies,  and  then  the  growth  settles  at  the  bottom 
of  the  tube. 

Agar. — At  brood-heat,  on  agar,  the  growth  is  quite  rapid,  and 
at  the  end  of  forty-eight  hours  gas  bubbles  have  formed  and  the 
growth  nearly  reached  the  surface. 

Bouillon. — Adding  glucose  to  the  bouillon  gives  a  medium  in 
which  an  abundant  growth  occurs. 

Cultivation  from  Spores. — Kitasato,  by  exposing  a  portion  of  sus- 
pected material  to  a  temperature  of  80°  C.  for  one  hour,  killed  off 
all  the  spores  save  those  of  tetanus,  which  were  then  cultivated. 

Staining. — All  the  ordinary  stains,  Gram's  method  also;  the 
spores  being  colored  in  the  usual  way. 

Pathogenesis. — A  small  amount  of  the  pure  culture  injected 
under  the  skin  of  experiment  animals  will  cause,  in  two  to  three 
days,  death  from  true  tetanus,  the  tetanic  condition  starting 
from  the  point  of  infection.  At  the  autopsy  nothing  characteristic 
or  abnormal  is  found,  and  the  bacilli  have  disappeared,  except 
near  the  point  of  entrance.     This  fact  is  explained  as  follows : 

Several  toxic  products  have  been  obtained  from  the  cultures, 
and  they  are  produced  in  the  body,  and  give  rise  to  the  morbid 
symptoms.  These  have  been  isolated,  and  when  injected  singly 
cause  some  of  the  tetanic  symptoms.  The  virus  enters  the 
circulation,  but  does  not  remain  in  the  tissues.  The  spores  are 
very  resistant  to  heat,  drying,  and  chemicals. 

Four  toxins  (among  them  tetanin,  tetanotoxin,  and  spasmo- 
toxin) have  been  found.  The  blood  and  the  urine  contain  the 
toxin  and  are  fatal  to  animals. 

Immunity. — Kitasato,  by  inoculation  of  sterilized  cultures,  has 
caused  immunity  to  the  effects  of  virulent  bacilli. 

An  anti-toxin  obtained  by  Tizzoni  and  Cattani  from  the  serum 
of  animals  made  immune  by  sterilized  cultures  has  been  used 


150 


ESSENTIALS   OF   BACTERIOLOGY. 


with  curative  effects  in  several  cases  of  tetanus  in  man.  It  is 
a  globulin,  but  differs  from  the  anthrax  anti-toxin,  and  it  is 
found  exclusively  in  the  serum.  By  precipitation  with  alcohol 
and  drying  in  vacuo  the  anti-toxin  is  obtained  in  a  solid  state. 
The  aqueous  solution  is  used  for  injection  subcutaneously  or 
subdurally  through  a  trephine  opening.  Its  injection  into  the 
spinal  canal  by  lumbar  puncture  has  also  been  recommended. 
Anti-toxin  is  more  beneficial  in  chronic  cases  than  in  acute. 

Habitat. — The  bacillus  is  present  in  garden  earth,  in  manure ; 
and  it  has  been  isolated  even  from  mortar. 

The  earth  of  special  districts  seems  to  contain  the  bacilli  in 
greater  quantities. 

Fig.  84. 


Bacillus  of  malignant  oedema,  from  the  body-juice  of  a  guinea-pig  inoculated  with 
garden  earth  ;  X  1000  (Frankel  and  Pfeiffer). 

Bacillus  (Edematis  Maligni.  (Koch,  1881.)  Vibrion  Septique. 
(Pasteur,  1875.) 

Origin. — In  garden  earth,  found  lately  also  in  man,  in  severe 
wounds  when  gangrene  with  oedema  had  developed.  Identical 
with  the  bacillus  found  in  Pasteur's  septicaemia. 

Form. — Rods  somewhat  smaller  than  the  anthrax  bacilli,  the 


PATHOGENIC    BACTERIA. 


151 


ends  rounded  very  sharply.    Long  threads  are  formed.    Very 
large  spores  which  cause  the  rods  to  become  spindle-shaped. 


Fig.  85. 


Fig.  86. 


ESfr-wl 


Cultures  in  agar  of  malignant 
(Edema,  after  24  hours,  at  37°  C. 
(Fr&nkel  and  Pfeiffer.) 


Bacillus  of  malignant  oedema 
growing  in  glucose-gelatine 
(Frankel  and  Pfeiffer). 


Properties. — Very  motile ;   liquefy  gelatine  ;   do  not  produce 
any  foul  gaseous  products  in  the  body. 


152        ESSENTIALS  OF  BACTERIOLOGY. 

Growth. — Grows  rapidly,  but  only  when  the  air  is  excluded, 
and  best  at  brood  or  body  heat. 

Roll  Cultures. — (After  Esmarch's  method.)  Small,  round  colo- 
nies with  fluid  contents,  under  low  power,  a  mass  of  motile 
threads  in  the  centre,  and  at  the  edges  a  wreath-like  border. 

High  Stab- Culture. — With  glucose  gelatine,  the  growth  at  first 
seen  in  the  bottom  of  the  tube,  with  a  general  liquefaction  of 
the  gelatine,  gases  develop  and  a  somewhat  unpleasant  odor. 

Agar. — The  gases  develop  more  strongly  in  this  medium,  and 
the  odor  is  more  prominent. 

Guinea-Tig  Bouillon. — In  an  atmosphere  of  hydrogen  cloud- 
ing of  the  entire  culture  medium  without  any  flocculent  pre- 
cipitate until  third  day. 

Staining.— Are  stained  with  the  ordinary  dyes,  but  Gram's 
method  is  not  applicable. 

Pathogenesis. — When  experiment  animals,  mice  or  guinea- 
pigs,  are  injected  with  a  pure  culture  under  the  skin  they  die  in 
8  to  15  hours,  and  the  following  picture  presents  itself  at  the 
autopsy  :  In  guinea-pigs  from  the  point  of  infection,  spreading 
over  a  large  area,  an  oedema  of  the  subcutaneous  tissues  and 
muscles,  which  are  saturated  with  a  clear  red  serous  exudate 
free  from  smell,  containing  great  quantities  of  bacilli. 

The  spleen  is  enlarged,  especially  in  mice.  The  bacilli  are 
not  found  in  the  viscera,  but  are  present  in  great  numbers  on 
the  surface,  i.  e.,  in  the  serous  coverings  of  the  different  orgfcns  ; 
though  when  any  length  of  time  has  elapsed  between  the  death 
of  the  animal  and  the  examination,  they  can  be  found  in  the 
inner  portions  of  the  organs,  for  they  grow  well  upon  the  dead 
body.  In  man  they  have  been  found  in  rapidly  spreading  gan- 
grene. They  are  present  in  the  soil,  in  putrefactions  of  various 
kinds,  and  in  dirty  water. 

Immunity. — Is  produced  by  injection  of  the  sterilized  cul- 
tures, and  also  the  filtered  bloody  serum  of  animals  dead  with 
the  disease. 

Spirillum  of  Relapsing  Fever.    (Obermeier.) 

Syn. — Spirochseta  Obermeieri. 

Origin. — Found  in  the  blood  of  recurrent  fever  patients, 
described  in  1873. 


PATHOGENIC   BACTERIA.  153 

Form.— Long,  wavy  threads  (16  to  40  fx  long),  a  true  spiril- 
lum ;  flagella  are  present. 

Properties. — Very  motile.     Has  not  been  cultivated. 

Staining.  —  Ordinary  aniline  stains.  Bismark  brown  best  for 
tissue  sections. 

Pathogenesis.  —Found  in  the  organs  and  blood  of  recurrent 
fever.  Man  and  monkeys  inoculated  with  blood  from  one  suf- 
fering from  this  disease  become  attacked  with  the  fever,  and 
in  their  blood  the  spirillum  is  again  found.  It  is  found  in  the 
blood,  only  in  the  relapses  (during  the  fever).  After  the  attack 
the  spirilla  gather  in  the  spleen  and  gradually  die  there.  It 
has  been  found  in  the  brain,  spleen,  liver,  and  kidneys.  In  the 
secretions  it  has  not  been  discovered. 


©°©« 


Fio.  87. 
.© 


<&Pod&      ° 


Spirochseta  Obermeieti  in  the  blood  (von  Jaksch). 

Bacillus  of  Soft  Chancre.    (Ducrey-Unna.) 

A  diplobacillus  which  is  specific  has  been  described  by  Ducrey 
as  obtained  from  the  secretion  and  in  the  depth  and  margins 
of  the  chancroid.  Unna's  bacillus  is  narrower  and  unbroken 
in  the  center. 

Cultivation. — Unsuccessful. 

Staining.— With  borax,  methylen-blue,  decolorized  with  weak 
acetic  acid. 

Pathogenesis. — Probably  a  mixed  infection  occurs  in  most 
chancroids,  especially  if  buboes  result.    The  bacillus  of  Ducrey 


154 


ESSENTIALS   OF   BACTERIOLOGY. 


is  not  found  in  unopened  buboes,  though  often  contaminating 
the  ulcerated  ones. 
Bacillus  Icteroides.    (Sanarelli,  1897.) 


Fig.  88. 


Considered  the  cause  of  yellow  fever  by  Sanarelli,  but  Stern- 
berg and  Novy  regard  this  as  not  determined.  It  is  not 
identical,  as  was  once  supposed,  with  the  Bacillus  X  of  Stern- 
berg, which  is  a  variety  of  the  Bacillus  Coli. 

Origin. — In  the  tissues  and  blood  of  yellow-fever  patients. 

Form. — A  small  bacillus  with  rounded  ends,  often  arranged  in 
pairs,  sometimes  in  threads,  with  lateral  flagella. 

Properties. — Motile,  readily  stained,  decolorized  by  Gram's 
method.  Does  not  liquefy  gelatine -nor  produce  glucose  fermen- 
tation.    Aerobic.     No  acid  reaction  in  milk. 

Growth. — Gelatine  plates ;  white  kidney -shaped  colonies,  with 
a  central  darker  portion  or  nucleus. 

Agar. — Colonies  look  like  drops  of  paraffin,  with  margins 
raised  above  the  surface.  Kept  alternately  at  22°  C.  and  37°  C, 
the  colonies  take  on  a  characteristic  appearance,  as  if  an  im- 
pression had  been  made  in  soft  wax. 


PATHOGENIC   BACTERIA.  155 

Potato. — Creamy  pale  growth,  turning  brown  in  a  week. 

Pathogenesis. — Dogs  and  rabbits,  when  inoculated  with  pure 
cultures,  are  affected  with  symptoms  exactly  similar  to  those 
seen  in  yellow-fever  patients— a  hemorrhagic  gastro-enteritis, 
steatosis  of  the  liver,  and  albuminuria. 

Two  theories  exist  as  to  the  etiology  of  yellow  fever :  (a)  That 
it  is  due  to  the  Bacillus  icteroides.  The  results  of  Sanarelli 
have  not  been  universally  accepted,  (b)  That  the  causative 
agent  is  so  small  as  to  be  microscopically  invisible,  and  that  it 
is  transmitted  from  man  to  man  through  the  bite  of  a  mos- 
quito, the  Stegomyia  fasciata,  which  acts  as  intermediate  host. 
Individuals  who  have  allowed  themselves  to  be  bitten  by  in- 
fected mosquitoes  have  contracted  the  disease,  while  others 
exposed  to  infection  in  all  ways  but  this  have  remained  well. 

Bacillus  of  Bubonic  Plague.     (Yersin  and  Kitasato,  1894. ) 

Bubonic  plague  or  pest  is  an  extremely  infectious  disease 
more  or  less  common  in  China  and  the  East,  and  is  believed 
to  have  its  origin  in  man  from  rats  and  other  rodents.  It 
spreads  with  great  rapidity,  especially  among  those  living  under 
unsanitary  conditions. 

Nearly  at  the  same  time  Yersin  and  Kitasato,  working  inde- 
pendently, discovered  in  the  bubonic  swellings  and  blood  of 
affected  persons  a  distinctive  bacillus  which  has  conformed  to 
all  the  conditions  necessary  to  make  it  the  cause  of  the  disease. 

Origin. — In  the  tissues  and  all  the  body  fluids  and  secretions 
of  affected  individuals. 

Form. — Short,  thick  rods  with  an  indistinct  capsule,  rounded 
ends.     Growing  in  chains  in  fluid  media. 

Properties. — Immotile.  Stains  readily.  No  spores.  Culti- 
vated best  in  oxygen,  but  is  facultative  anaerobic.  Stains 
stronger  at  the  ends,  producing  bipolar  appearance.  Gelatine 
not  liquefied.     Easily  destroyed  by  sunlight  and  drying. 

Growth.— Best  at  37°  C. 

Gelatine. — At  22°  C,  in  24  hours  white,  point-like  colonies  on 
the  plates,  with  broad  and  flat  surface,  turning  gray  and  then 
brown. 

Stab. — Snow-white,  spreading  out  on  the  surface  to  the  edge, 
and  fluorescent. 


156 


ESSENTIALS  OF  BACTERIOLOGY. 


Bouillon. — Granular  precipitate,  with  clear  fluid  above. 

Agar  and  Blood-serum. — Glass-like  colonies  like  drops  of  dew 
at  first,  then  growing  larger  with  iridescent  edges. 

Potato. — At  37°  C.  small  white  mass. 

No  gas  formation  in  glucose  media. 

Staining  readily  with  all  basic  dyes. 

Pathogenesis. — After  subcutaneous  injection  in  rats  death  fol- 
lows in  40  to  60  hours,  with  symptoms  of  severe  toxaemia  and 
convulsions.  The  point  of  infection  shows  a  local  oedema  and 
inflammation  of  the  lymphatics.  All  the  organs  congested  and 
surrounded  by  a  bloody  exudate.  The  characteristic  bacilli  in 
all  the  tissues  and  secretions.     Nearly  all  the  domestic  animals 

Fig.  89. 


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Bacillus  of  bubonic  plague  (Yersin). 


are  susceptible.  Mosquitoes  and  pigeons,  however,  are  im- 
mune; flies  are  not. 

Products. — A  toxin  has  been  obtained  and  immunity  has  been 
effected;  the  serum  of  immune  animals  has  protective  prop- 
erties. The  serum  likewise  shows  agglutinating  powers,  as  with 
typhoid  and  cholera  serums. 

Habitat.— Not  found  in  water,  but  most  likely  spreads  from  the 
soil  in  damp  and  darkened  areas.     Rats  become  affected  first, 


PATHOGENIC    BACTERIA.  157 

and  then  through  bites  and  scratches  affect  man  and  other  ani- 
mals. Clothing,  vomit,  and  the  excretions  generally,  likewise, 
act  as  carriers  of  the  infection.  In  man  three  forms  of  the  dis- 
ease are  recognized  according  to  the  mode  of  infection  and 
course  of  the  disease — viz.,  bubonic,  pulmonic,  septicemic. 

Bacillus  Dysenteriae.    (Shiga,  1898.) 

The  term  dysentery  is  applied  to  an  intestinal  disease  dis- 
playing more  or  less  constancy  in  its  clinical  manifestations, 
but  having,  as  is  now  known,  a  variety  of  causative  agents.  It 
is  fairly  certain  that  one  type  is  the  result  of  infection  with  an 
amoeba,  while  non-amoebic  forms  can  probably  be  produced  by 
several  bacteria.  Chief  among  these  is  the  bacillus  first  de- 
scribed by  Shiga  in  Japan,  and  since  then  found  by  Kruse  in 
Germany,  by  Flexner,  Strong,  and  Harvie  in  the  Philippine 
Islands,  and  by  Vedder  and  Duval  in  the  United  States. 
Although  it  is  not  absolutely  proved  that  it  is  the  cause  of  the 
disease,  still  the  feet  that  it  is  constantly  present  in  the  faeces  in 
one  type  of  dysentery,  that  such  cases  give  a  positive  agglu- 
tination reaction,  the  production  of  a  curative  serum  by  the 
immunization  of  animals  with  pure  cultures,  and  the  results 
on  experiment  animals,  leave  little  doubt  as  to  the  specificity 
of  the  organism. 

Origin. — The  dejecta  of  dysenteric  patients. 

Form. — A  plump  bacillus  with  rounded  ends,  resembling  the 
typhoid  and  colon  bacilli. 

Properties. — Motility  doubtful,  but  numerous  flagella  have 
been  demonstrated.     Does  not  form  spores. 

Staining. — Stains  readily,  negative  to  Gram,  facultative  an- 
aerobe. 

Growth.— Best  at  37°  C.  Killed  by  ten  minutes'  exposure  to 
55°  C. 

Gelatine. — A  white  line  of  growth  along  puncture;  super- 
ficial growth  slight. 

Bouillon. — Uniform  clouding.  Indol  usually  not  produced; 
milk  not  coagulated. 

Agar. — Resembles  typhoid  bacillus. 

Potato. — Thin  whitish  layer,  turning  light  brown. 

No  gas-formation  in  glucose  or  lactose  media. 


158        ESSENTIALS  OP  BACTERIOLOGY. 

Pathogenesis. — Mice  and  guinea-pigs  die  in  one  or  two  days 
after  intraperitoneal  inoculation.  Rabbits  usually  recover, 
though  lesions  analogous  to  those  of  human  dysentery  have 
been  produced.  Dogs  die  in  five  or  six  days,  with  well-marked 
diarrhoea. 

Products. — The  patient's  blood-serum  agglutinates  the  bacillus 
in  cases  in  which  it  can  be  cultivated  from  the  stools.  The  re- 
action is  absent  from  other  cases.  Shiga  has  reduced  the  mor- 
tality from  34.7  to  9  per  cent,  by  means  of  a  serum  obtained 
from  immunized  horses. 

Habitat. — Found  in  the  stools  and  in  shreds  of  mucous  mem- 
brane from  the  intestinal  walls. 

Bacillus  Aerogenes  Capsulatus.     (Welch,  1891.) 

Origin. — The  intestine  of  man  and  animals,  soil,  sewage,  and 
water. 

Form. — A  thick  bacillus,  3  to  6  \i  in  length,  frequently  capsu- 
lated. 

Properties. — Not  motile,  anaerobic,  forms  spores  chiefly  in  cul- 
tures on  blood-serum. 

Growth.— Best  at  37°  C. 

Gelatine. — Liquefied  slowly  or  not  at  all. 

Bouillon. — Forms  gas. 

Milk. — Coagulated  and  becomes  acid. 

Potato. — Thin,  grayish-white  growth  with  gas-production. 

Forms  gas  in  abundance  on  dextrose,  lactose,  or  saccharose 
media. 

Pathogenesis. — Is  not  usually  pathogenic  for  rabbits  and  mice, 
though  in  guinea-pigs  and  birds  it  produces  "gas  phlegmons." 
It  is  sometimes  found  in  autopsies  on  human  subjects,  produc- 
ing bubbles  or  cavities  in  the  viscera  (Schaumorgane),  but  this 
is  probably  due  to  postmortem  migration  of  the  germ  from  the 
intestine.  It  has  been  recovered  from  the  blood  during  life, 
however,  and.  is  the  most  frequent  cause  of  emphysematous 
gangrene.  Various  foreign  observers  have  described  organisms 
having  similar  properties  and  have  given  them  such  names  as 
Bacillus  perfringens,  Bacillus  enteritidis,  Granulobacillus  immo- 
bilis,  etc.,  but  they  were  probably  dealing  with  the  Bacillus 
aerogenes  capsulatus. 


PATHOGENIC    BACTERIA.  159 

Micrococcus  Melitensis.    (Bruce,  1887.) 

Malta  fever,  also  known  as  Mediterranean  fever,  occurs  in  the 
region  from  which  it  derives  its  name,  but  has  been  observed  in 
India,  the  Philippine  Islands,  and  Porto  Rico.  Bruce  culti- 
vated a  micrococcus  from  the  spleen  and  proved  its  specificity. 

Origin. — Is  found  most  abundantly  in  the  spleen. 

Form. — Rounded  or  oval,  5  //  in  diameter,  singly,  in  pairs,  or 
short  chains. 

Properties. — Non-motile,  though  flagella  said  to  be  present; 
grows  slowly,  best  at  body-temperature. 

Gelatine. — Not  liquefied ;  growth  very  slow. 

Bouillon. — Turbid,  with  sediment. 

Agar. — Pearly  white  growths. 

Potato. — Slight  invisible  growth. 

Stained  by  ordinary  aniline  dyes. 

The  disease  may  be  produced  in  monkeys  by  even  small 
amounts  of  pure  culture.  In  man  a  chronic,  remittent  febrile 
disease  is  produced,  with  sweating  and  arthritis.  The  mortality 
is  2  per  cent.  A  serum  reaction  can  be  obtained  and  is  diag- 
nostic. 

Micro-organisms  have  been  found  by  various  observers  in 
measles,  scarlatina,  mumps,  and  whooping-cough,  but  their 
specificity  is  still  in  doubt. 

PATHOGENIC  PROTOZOA. 

Certain  diseases  are  produced  by  animal  parasites  belonging 
to  the  protozoa,  and  although  not  pertaining  to  the  realm  of 
bacteriology,  still  the  fact  that  they  were  long  considered  bac- 
terial in  nature  and  require  somewhat  similar  methods  for  their 
study  renders  it  proper  to  include  a  brief  mention  of  them. 

The  Malarial  Parasite.  It  has  been  definitely  proved  that 
malarial  fever  is  the  result  of  the  presence  in  the  blood  of  a 
protozoon  which  in  the  vast  majority  of  cases  gains  entrance 
to  the  body  through  the  bite  of  a  particular  genus  of  mosquito 
(Anopheles).  Three  varieties  of  the  organism  are  recognized 
in  man,  though  possibly  more  exist,  and  each  produces  a  char- 
acteristic clinical  picture.  1.  The  Hazmamazba  vivax,  the  para- 
site of  tertian  fever.     2.  The  Hmmamazba  malarias,  the  parasite 


160  ESSENTIALS    OP    BACTERIOLOGY. 

of  quartan  fever.     3.  The  Hcemomenas  prsecox,  the  parasite  of 
sestivo-autumnal  fever. 

According  to  its  situation,  the  parasite  exhibits  two  distinct 
phases  of  existence :  in  the  human  blood  it  passes  through  an 
asexual  reproductive  cycle,  while  in  the  body  of  the  mosquito 
it  undergoes  an  entirely  different  series  of  sexually  reproductive 
changes.  It  is  simpler  first  to  describe  the  life  history  of  the 
organism  in  general,  pointing  out  the  differences  shown  by  the 
three  varieties  later. 

1.  The  Asexual  Cycle  in  Man. — An  infected  mosquito  conveys 
the  parasites  into  the  blood  as  minute  hyaline  bodies  which 
enter  the  blood-cells.  At  first  they  are  small,  round,  colorless 
bodies,  exhibiting  more  or  less  active  amoeboid  motion  in  the 
fresh  blood.  Sometimes,  particularly  in  the  sestivo-autumnal 
form,  a  ring  shape  is  assumed.  Their  size  gradually  increases 
and  pigment  granules  appear,  while  in  stained  specimens  a 
nucleus  containing  chromatin  granules  is  visible.  As  the  para- 
site approaches  maturity  the  chromatin  becomes  scattered, 
and  finally  the  protoplasm  divides  into  six  to  twenty  spores 
(merozoites),  each  containing  a  portion  of  the  chromatin.  The 
number  of  spores  formed  and  their  arrangement  before  seg- 
mentation takes  place  differ  in  the  three  varieties  and  will  be 
noted  below.  The  spores  burst  through  the  envelop  of. the  red 
corpuscle  and  become  free  in  the  blood,  but  speedily  enter  fresh 
corpuscles  and  pass  through  the  same  series  of  changes.  The 
febrile  stage  is  synchronous  with  sporulation  and  liberation  of 
the  young  forms. 

Certain  of  the  parasites  do  not,  however,  go  on  to  segmenta- 
tion, but,  after  reaching  maturity,  remain  quiescent  and  form 
the  so-called  gametes  or  sexual  types.  In  the  tertian  and  quartan 
varieties  these  are  not  very  different  from  the  mature  organ- 
isms, but  the  sestivo-autumnal  gametes  are  crescentic  in  shape 
and  very  characteristic. 

2.  The  Sexual  Cycle  in  the  Mosquito. — If,  now,  the  blood  is  shed, 
certain  of  the  gametes  (the  male  forms  or  microgametocytes)  ex- 
trude long  protoplasmic  processes  containing  a  central  core  of 
chromatin,  and  which  represent  the  male  fertilizing  element 
(microgametes).    These  become  detached,  and,  entering  a  female 


Plate    III. 

Various  Forms  of  Malarial  Parasites  (Thayer  and  Hewetson).  Figs.  1-10 
inclusive,  tertian  organisms ;  Figs.  11-17  inclusive,  quartan  organisms ;  Figs. 
18-27  inclusive,  estivo-autumnal  organisms. 

Fig.  1.— Young  hyaline  form;  2,  hyaline  form  with  beginning  pigmenta- 
tion ;  3,  pigmented  form ;  4,  full-grown  pigmented  form  ;  5,  6,  7,  8,  segmenting 
forms  ;  9,  mature  pigmented  form  ;  10,  flagellate  form. 

Fig.  11.— Young  hyaline  form  ;  12,  13,  pigmented  forms ;  14,  fully  developed 
form  ;  15,  16,  segmenting  forms  ;  17,  flagellate  form. 

Figs.  18, 19,  20.— Ring-like  and  cross-like  hyaline  forms:  21,  22,  pigmented 
forms  ;  23,  24,  segmenting  forms  ;  25,  26,  '27,  crescents. 


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PATHOGENIC    BACTERIA.  161 

gamete  (macrogamete),  a  true  sexual  fertilizing  process  takes 
place.  In  the  alimentary  canal  of  the  mosquito  these  fertilized 
cells  penetrate  the  stomach-walls  and  form  cysts  filled  with  a 
large  number  of  filiform  spores,  which  are  extruded  into  the 
body  cavity  of  the  insect,  and  some  of  which  reach  the  salivary 
glands,  whence  they  are  ejected  when  the  mosquito  bites. 
This  cycle  of  development  takes  seven  or  eight  days. 
Differential  points  of  the  three  forms : 

1.  The  Tertian  Form. — The  adult  forms  are  large,  not  very  re- 
fractile,  and  their  outline  is  somewhat  indistinct.  There  is  an 
abundance  of  fine  pigment-granules,  and  the  ameboid  motion 
is  vigorous.  Segmenting  forms  divide  into  15  to  20  merozoites ; 
the  sexual  forms  or  gametes  are  large.  The  red  cell  containing 
the  organism  is  swollen  and  pale.  Sporulation  and,  therefore, 
the  malarial  paroxysm  occur  every  forty-eight  hours. 

2.  The  Quartan  Form. — The  organism  is  smaller,  is  more  re- 
fractile,  and  its  outline  is  more  distinct.  The  pigment  is  coarse 
and  situated  at  the  periphery  of  the  organism,  while  the  proto- 
plasmic motion  is  sluggish.  Segmentation  forms  only  6  to  12 
spores,  and  has  the  regular  "  daisy-head "  appearance ;  the 
gametes  are  small.  The  red  cells  become  dark  in  color,  and 
the  cycle  requires  seventy-two  hours. 

3.  JEstivo-autumnal  Form. — The  adult  forms  are  found  mainly 
in  the  spleen  and  other  viscera,  and  do  not  very  often  occur  in 
the  peripheral  blood ;  their  outline  is  sharp,  and  they  are  highly 
refractile.  The  pigment  is  scanty  and  fine;  the  motion  is 
active.  A  variable  number  of  merozoites  is  formed — usually  6 
to  12.  The  gametes  are  characteristic,  being  crescentic  in 
shape  and  very  resistant  to  quinine.  The  red  cell  becomes 
shrivelled  and  yellowish.  The  cycle  usually  takes  forty-eight 
hours,  though  it  is  somewhat  variable. 

Mixed  infections  with  the  different  organisms  or  with  two  or 
more  broods  of  the  same  organism  may  occur,  so  that  quotidian 
and  irregular  paroxysms  may  be  produced. 

Methods  of  Examination. 

1.  Fresh  preparations  are  made  by  placing  a  small  drop  of 
blood  on  a  slide  and  a  cover-glass  over  it,  so  that  only  a  thin 
film  is  formed.  A  ring  of  vaseline  is  smeared  over  the  edges 
11 


162  ESSENTIALS    OF    BACTERIOLOGY. 

of  the  cover-glass  to  prevent  evaporation.  This  is  the  best 
method  for  studying  flagellation  and  fertilization,  but  is  less 
satisfactory  for  routine  clinical  work  than — 

2.  Stained  Smears. — These  are  made  by  spreading  a  drop  of 
blood  in  a  thin  film  over  one  slide  with  the  edge  of  another, 
drying  in  the  air,  and  staining.  Many  stains  have  been  devised 
for  the  malarial  organism,  but  the  following  are  sufficient  for 
ordinary  use : 

(1)  Marchoux's  Thionin  Stain.— Add  20  c.c.  of  saturated  solu- 
tion of  thionin  in  50  per  cent,  alcohol  to  100  c.c.  of  2  per  cent, 
carbolic  acid.  Fix  the  smears  and  stain  for  fifteen  to  twenty 
seconds.  The  malarial  organisms  are  stained  a  deep  purple, 
strongly  contrasting  with  the  faint  green  of  the  red  cells,  so 
that  they  are  readily  recognized. 

(2)  Jenner's  Stain. — This  is  excellent  for  routine  work,  as  no 
preparatory  fixation  is  required.  Equal  parts  of  a  1.2  per  cent, 
aqueous  solution  of  Griibler's  water-soluble  eosin  and  a  1  per 
cent,  aqueous  solution  of  Griibler's  medicinal  methylin-blue 
are  mixed  and  the  resulting  precipitate  allowed  to  stand  for 
twenty-four  hours,  washed,  and  dried.  Half  a  gram  of  this  is 
dissolved  in  100  c.c.  of  pure  methyl-alcohol.  The  smears  are 
dropped  into  this  stain  for  one  to  three  minutes,  without  pre- 
vious fixation,  and  at  once  rinsed  in  distilled  water.  The 
malarial  parasites  are  stained  blue,  the  cell-bodies  a  reddish 
brown. 

(3)  Wright's  Chromatin  Stain. — This  is  the  best  of  the  chroma- 
tin stains.  For  its  preparation,  which  is  quite  complicated,  see 
Wright,  Journal  of  Medical  Research,  vol.  vii.,  1902.  It  is  used 
as  follows : 

1.  The  stain  is  poured  over  the  film  and  allowed  to  remain 
for  one  minute  to  secure  fixation. 

2.  Add  distilled  water  drop  by  drop  until  a  metallic  scum  is 
formed  on  the  surface.  The  staining  now  takes  place  and  re- 
quires two  to  three  minutes.  Wash  in  distilled  water  until  a 
pinkish  tint  appears  in  the  thin  portions  of  the  smear.  The 
body  of  the  malarial  parasite  is  stained  blue,  and  its  chromatin 
a  lilac  to  red  color.    The  red  cells  are  orange  pink. 


PATHOGENIC    BACTERIA.  163 

If  possible,  examinations  for  malarial  organisms  should 
always  be  made  before  quinine  is  administered. 

Amoeba  Bysenteriae.  Found  in  the  intestinal  ulcers,  fasces, 
and  secondary  liver  abscesses  in  certain  cases  of  dysentery.  A 
non-pathogenic  form,  Amoeba  coli,  also  occurs.  The  Amoeba 
dysenteries  is  a  unicellular  animal  organism  measuring  25  to 
35  fi  in  diameter,  though  larger  and  smaller  forms  occur.  There 
are  a  nucleus  and  a  nucleolus ;  the  protoplasm  of  the  cell-body 
is  vacuolated  and  often  contains  red  blood-cells  and  bacteria. 
In  fresh,  warm  stools  active  ameboid  motion  may  be  observed. 
The  non-pathogenic  form  is  smaller  and  never  contains  red 
blood-cells. 

Small-pox  and  Vaccinia.  The  exciting  agent  of  small-pox  is 
still  unknown,  but  numerous  bacteria  and  protozoon-like  bodies 
have  been  described  and  given  etiological  significance  by  vari- 
ous authors.  There  is  some  evidence  in  favor  of  Funck's  belief 
that  vaccinia  is  caused  by  a  protozoon,  the  Sporidium  vaccinale. 
Animals  inoculated  with  this  organism  developed  both  vaccinia 
and  variola. 

Trypanosomes.  These  are  protozoa  belonging  to  the  order 
flagettaJta,  and  have  been  found  in  the  blood  in  certain  diseases 
of  man  and  animals.  Surra,  a  fatal  tropical  disease  of  horses 
and  mules,  the  tse-tse  fly  disease  of  South  Africa,  and  the 
sleeping  sickness  or  negro  lethargy  of  the  Guinea  coast,  are  due 
to  organisms  of  this  group.  It  is  probable  that  insects  of  vari- 
ous species  are  the  intermediary  or  definitive  hosts  of  the 
trypanosome  and  convey  the  infection  by  their  bites. 

Texas  cattle-fever  or  bovine  malaria  is  due  to  an  endoglobular 
parasite,  the  Pi/rosoma  bigwninum,  not  unlike  the  malarial 
organism,  and  is  transmitted  through  the  larvae  of  the  cattle- 
tick.  A  similar  organism  has  lately  been  found  to  be  the  cause 
of  the  Rocky  Mountain  fever  of  man.  The  infection  here  also 
seems  to  be  through  the  mediation  of  a  tick. 


164        ESSENTIALS  OF  BACTERIOLOGY. 

CHAPTER  IV. 

BACTERIA  PATHOGENIC  FOR  ANIMALS  BUT  NOT  FOR  MAN. 

Bacillus  of  Symptomatic  Anthrax.     (Bollinger  and  Feser.) 

(Charbon  symptomatique.     Arloing,  Cornevin,  and  Thomas.; 

Origin. — This  bacillus,  described  already  in  1879,  has  only 
lately  been  isolated,  and  by  animal  inoculation  shown  to  be  the 
cause  of  the  "  black-leg"  or  "  quarter  evil"  disease  of  cattle. 

Form. — Large  slender  rods,  which  swell  up  at  one  end  or  in 
the  middle  for  the  spore.     (See  Plate  IV.,  Fig.  1.) 

Properties. — They  are  motile,  and  liquefy  gelatine  quite 
rapidly. 

A  rancid  odor  is  developed  in  the  cultures. 

Cultures.— The  growth  occurs  slowly,  and  only  in  an  atmo- 
sphere of  hydrogen,  being  very  easily  destroyed  by  oxygen  and 
carbon  dioxide  ;  grows  best  at  blood  heat ;  under  15°  C.  no 
growth. 

Glucose-gelatine.— In  a  few  days  little  round  colonies  develop, 
which,  under  low  power,  show  hairy  processes  around  a  compact 
centre. 

Stab  Cultures  in  full  test  tubes. — The  first  growth  in  the  lower 
portion  of  the  tube  not  very  characteristic.  Gases  develop 
after  a  few  days,  and  the  gelatine  becomes  liquid. 

Agar  at  brood  temperature,  in  24  to  48  hours,  an  abundant 
growth  with  a  sour  odor  and  abundant  gas  formation. 

Staining.— Ordinary  methods.  Gram's  method  is  not  appli- 
cable to  the  rods ;  but  the  spores  can  be  colored  by  the  regular 
double  stain  for  spores. 

Pathogenesis. — If  a  small  amount  of  the  culture  be  injected 
under  the  skin  of  a  guinea-pig,  in  twenty  hours  a  rise  of  tempera- 
ture, pain  at  the  site  of  injection,  and  in  a  few  hours  more 
death.  At  the  autopsy,  the  tissues  blackened  in  color  and 
soaked  with  a  bloody  serous  fluid  ;  in  the  connective  tissue  large 
collections  of  gas,  but  only  in  the  neighborhood  of  the  point 
of  infection.     The  bacilli  are  found  in  great  numbers  in  the 


BACTERIA    PATHOGENIC    FOR    ANIMALS 


165 


serum,  but  only  appear  in  the  viscera  some  time  after  death, 
when  spores  have  developed. 

The  animals  are  usually  infected  through  wounds  on  the 
extremities  ;  the  stalls  or  meadows  having  been  dirtied  by  the 
spore-containing  blood  of  animals  previously  dead  of  the  dis- 
ease. " Baaachbrand"  is  the  German  name;  "  Cliarbon  symp- 
tomatique,"  the  French,  from  the  resemblance  in  its  symptoms 
to  anthrax. 

Immunity.—  Rabbits,  dogs,  pigs,  and  fowls  are  immune  by 
nature,  but  if  the  bacilli  are  placed  in  a  20  per  cent,  solution  of 
lactic  acid,  and  the  mixture  injected,  the  disease  develops  in 
them.  The  lactic  acid  is  supposed  to  destroy  some  of  the 
natural  resistance  of  the  animal's  cells. 

When  a  bouillon  culture  is  allowed  to  stand  a  few  days,  the 
bacilli  therein  lose  their  virulence,  and  animals  are  no  longer  af- 
fected by  them. 

But  if  they  are  placed  in  20  per  cent,  lactic  acid  and  the  mix- 
ture injected,  their  virulence  returns. 

Immunity  is  produced  by  the  injections  of  these  weakened 
cultures,  and  also  by  some  of  the  products  which  have  been  ob- 
tained from  the  cultures. 

Bacillus  of  Chicken  Cholera.    (Pasteur.) 

Syn.— Micrococcus  cholera  gallinarum.  Microbe  en  huit.  Ba- 
cillus avicidus.     Bacillus  of  fowl  septicemia. 

Origin. — In  1879  Perroncito  observed  this  cocci-like  bacillus 
in  diseases  of  chickens,  and  Pasteur,  in  1880,  isolated  and 
reproduced  the  disease  with  the  microbe  in  question. 

Form. — At  first  it  was  thought  to  be  a  micro- 
coccus, but  it  has  been  seen  to  be  a  short  rod 
about  twice  as  long  as  it  is  broad,  the  ends 
slightly  rounded.  The  centre  is  very  slightly 
influenced  by  the  aniline  colors,  the  poles 
easily,  so  that  in  stained  specimens  the  bacil- 
lus looks  like  a  dumb-bell  or  a  figure-of-eight. 
(Microbe  en  huit. ) 

Properties. — They  do  not  possess  self-move- 
ment ;  do  not  liquefy  gelatine. 

Growth. — Occurs  at  ordinary  temperature,  requiring  oxygen 
for  development.     It  grows  very  slowly. 


Fig 


Chicken  cholera 
in  blood  1000  X- 
(Frankel  and 
Pfeiffer.) 


166        ESSENTIALS  OP  BACTERIOLOGY. 

Gelatine  Plates. — In  the  course  of  three  days  little  round, 
white  colonies,  which  seldom  increase  in  size,  having  a  rough 
border  and  very  finely  granulated. 

Stab  Culture. — A  very  delicate  gray  line  along  the  needle- 
track,  which  does  not  become  much  larger. 

Agar  Stroke  Culture.— A  moist,  grayish-colored  skin,  more 
appreciable  at  brood  heat. 

Potato.— At  brood  heat  after  several  days  a  very  thin,  trans- 
parent growth. 

Staining.  —  Methylin  blue  gives  the  best  picture.  Gram's 
method  is  not  applicable.  As  the  bacillus  is  easily  decolorized, 
aniline  oil  is  used  for  dehydrating  tissue  sections,  instead  of 
alcohol. 

Method  : 

Loffler's  methylin  blue  .  £  hour. 

Alcohol 5  seconds. 

Aniline  oil 5  minutes. 

Turpentine 1  minute. 

Xylol  and  Canada  balsam. 

Pathogenesis. — Feeding  the  fowls  or  injecting  under  the  skin 
will  cause  their  death  in  from  12  to  24  hours,  the  symptoms  pre- 
ceding death  being  those  of  a  heavy  septicaemia. 

The  bacillus  is  then  found  in  the  blood  and  viscera,  and  the 
intestinal  discharges,  the  intestines  presenting  a  hemorrhagic 
inflammation. 

Guinea-pigs  and  sheep  do  not  react.  Mice  and  rabbits  -are 
affected  in  the  same  manner  as  the  fowls. 

Immunity. — Pasteur,  by  injecting  different-aged  cultures  into 
fowls,  produced  in  them  only  a  local  inflammation,  and  they 
were  then  immune.  But  as  the  strength  of  these  cultures  could 
not  be  estimated,  many  fowls  died  and  the  healthy  ones  were 
endangered  from  the  intestinal  excretions,  which  is  the  chief 
manner  of  infection  naturally  ;  the  freces  becoming  mixed  with 
the  food. 

Bacteria  of  Hemorrhagic  Septicaemia.    (Hueppe.) 

Under  this  heading  Hueppe  has  gathered  a  number  of  bac- 
teria very  similar  to  the  bacillus  of  chicken  cholera,  differing 


BACTERIA   PATHOGENIC    FOR   ANIMALS.  167 

from  it  and  each  other  but  very  little.    They  have  been  described 
by  various  observers  and  found  in  different  diseases. 

(1)  The  bacteria  of  this  group  color  themselves  strongly  at 
the  poles,  giving  rise  to  tbe  dumb-bell  shape.  They  do  not  take 
the  Oram  stain.     They  are  without  spores, 

(2)  And  do  not  liquefy  gelatine. 

They  have  been  placed  in  three  general  divisions  : — 

f  Wild  Plague.     (Hueppe.) 

I  German  Swine  Plague.     (Loffler,  Schtitz.) 
1st  division.  -{  Rabbit  Septicaemia. 

I  Ox  Plague.     (Oresti-Armanni.) 

[  Steer  Plague.     (Kitt.) 
The  bacteria  of  the  first  division  are  not  motile,  do  not  grow- 
on  potato,  and  are  found  scattered  through  the  bloodvessels. 
A  local  reaction  is  uncommon. 

f  American  Swine  Plague.     (Billings.) 

J  French  Swine  Plague.    (CornilandChantemesse.) 
2d  division.  {  CaUle  piague      Texas  Je(jer>     (Billings.) 

{  Frog  Plague.     (E berth.) 

Here  the  bacteria  are  motile.  They  grow  on  potatoes  and 
are  similar  to  the  typhoid  bacillus  in  gelatine.  They  form 
small  embolic  processes  in  the  capillaries.  They  cause  only 
a  local  disturbance  in  rabbits  when  subcutnneously  injected. 
An  acid  fermentation  is  produced  in  milk. 
,.  .  .        f  Hog  Cholera.     (Salmon.) 

1  Swedish  Swine  Plague.     (Lelander.) 

The  bacteria  of  this  third  division  are  very  motile.  The  hog- 
cholera  bacilli  lie  in  the  spleen  and  other  organs  in  small  masses 
like  the  typhoid  bacillus. 

Rabbits  die  in  four  to  eight  days  without  any  local  disturb- 
ance.    The  growth  on  potato  is  strong. 

The  Swedish  swine-plague  bacillus  occupies  a  position  be- 
tween that  of  Hog  Cholera  and  Bacillus  Coli  Communis. 

The  various  swine-plague  bacilli  are  but  little  active  in  fowls, 
differing  thus  widely  from  the  chicken  cholera  bacillus. 

Bacillus  of  Erysipelas  of  Swine.  (Loffler,  Schiitz.)  Schweine- 
roilaufbacillus  (German).     Bouget  du  pore  (French). 


168        ESSENTIALS  OF  BACTERIOLOGY. 

Origin. — Found  in  the  spleen  of  an  erysipelatous  swine  by 
Loffler  in  1885. 

Form.— One  of  the  smallest  forms  of  bacilli  known  ;  very  thin, 
seldom  longer  than  1  p,  looking  at  first  like  little  needle-like 
crystals.     Spores  have  not  been  found. 

Properties. — They  are  motile  ;  do  not  liquefy  gelatine. 

Growth  in  culture  at  ordinary  temperature,  very  slowly,  and 
the  less  oxygen  the  better  the  growth. 

Gelatine  Plate. — On  third  day  little  silver-gray  specks,  seen 
best  with  a  dark  background,  coalescing  after  awhile,  pro- 
ducing a  clouding  of  the  entire  plate. 

Stab  Cultures. — In  a  few  days  a  very  light,  silvery -like  clouding, 
which  gradually  involves  the  entire  gelatine ;  held  up  against 
a  dark  object,  it  comes  plainly  into  view. 

Staining. — All  ordinary  dyes  and  Gram's  method  also. 

Tissue  sections  stained  by  Gram's  method  show  the  bacilli  in 
the  cells,  capillaries,  and  arterioles  in  great  numbers. 

Pathogenesis.  —Swine,  mice,  rabbits,  and  pigeons  are  sus- 
ceptible ;  guinea-pigs  and  chickens,  immune. 

When  swine  are  infected  through  food  or  by  injection  a  tor- 
pidity develops  with  diarrhoea  and  fever,  and  on  the  belly  and 
breast  red  spots  occur  which  coalesce,  but  do  not  give  rise  to 
any  pain  or  swelling.  The  animal  dies  from  exhaustion  in  24  to 
48  hours.     In  mice  the  lids  are  glued  together  with  pus. 

At  the  autopsy  the  liver,  spleen,  and  glands  are  enlarged  and 
congested,  little  hemorrhages  occurring  in  the  intestinal  mucous 
membrane  and  that  of  the  stomach. 

Bacilli  are  found  in  the  blood  and  all  the  viscera. 

One  attack,  if  withstood,  protects  against  succeeding  ones. 

Immunity. — Has  also  been  attained  b}T  injecting  vaccines  of 
two  separate  strengths. 

Bacillus  Murisepticus.     (Koch.)    Mouse  septicaemia. 

Origin.— Found  in  the  body  of  a  mouse  which  had  died  from 
injection  of  putrid  blood,  and  described  by  Koch  in  1878. 

Form.— Differs  in  no  particular  from  the  bacillus  of  swine 
erysipelas,  excepting  that  it  is  a  very  little  shorter,  making  it 
the  smallest  known  bacillus.  Spores  have  been  found,  the  cul- 
tures exactly  similar  to  those  of  swine  erysipelas. 


BACTERIA   PATHOGENIC   FOR   ANIMALS.  169 

The  pathological  actions  are  also  similar.  Field  mice  are 
immune  ;  whereas  for  house  and  white  mice  the  bacillus  is  fatal 
in  two  to  three  days. 

Micrococcus  of  Mai  de  Pis.  (Nocard.)  Gangrenous  mastitis 
of  sheep. 

Origin.— In  the  milk  and  serum  of  a  sheep  sick  with  the 
umai  depis." 

Form. — Yery  small  cocci  seldom  in  chains. 

Properties,  immotile  ;  liquefying  gelatine. 

Growth.—  Growth  occurs  best  between  20°  and  37°  C,  is  very 
rapid,  and  irrespective  of  oxygen. 

Plates  of  Gelatine.— White  round  colonies,  some  on  the  surface 
and  some  in  the  deeper  strata,  with  low  power,  appearing  brown 
surrounded  by  a  transparent  areola. 

Stab  Culture. — Very  profuse  along  the  needle-track,  in  the 
form  of  a  cone  after  two  days,  the  colonies  having  gathered  at 
the  apex. 

Potato. — A  dirty  gray,  not  very  abundant,  layer  somewhat 
viscid. 

Staining,  with  ordinary  methods  ;  also  Gram's  method. 

Pathogenesis.— If  a  pure  culture  is  injected  into  the  mammary 
gland  of  sheep,  a  u  mal  de  pis"  is  produced  which  causes  the 
death  of  the  animal  in  24  to  48  hours.  The  breast  is  found 
oedematous,  likewise  the  thighs  and  perineum  ;  the  mammae 
very  much  enlarged,  and  at  the  nipples  a  blue-violet  coloration. 
The  spleen  is  small  and  black  ;  other  animals  are  less  susceptible. 
In  rabbits  abscesses  at  the  point  of  infection,  but  no  general 
affection. 

Bacillus  Alvei.  (Cheshire  and  Cheyne.)  Bacillus  melittoph- 
tharus.     (Cohn.) 

Origin.—  In  foul-brood  of  bees. 

Form. — Slender  rods,  with  round  and  conical-pointed  ends ; 
very  large  oval  spores,  the  rod  becoming  spindle-shaped  when 
they  appear. 

Properties. — Motile,  liquefying  gelatine  rapidly. 

Grotvth.— Grows  best  between  20°  C.  and  37°  C,  very  slowly  ; 
aerobic. 

Gelatine  Plates.— Small  grooves  are  slowly  formed,  which  unite 


170  ESSENTIALS    OF   BACTERIOLOGY. 

so  as  to  form  a  circle  or  pear-shaped  growth,  from  which  linear 
grooves  again  start. 

Stab  Culture.— Grows  first  on  surface,  then  gradually  along 
the  needle-track,  long  processes  shooting  out  from  the  same, 
clouding  the  gelatine.  Later,  air-buhbles  form  like  the  cholera 
culture,  and  in  two  weeks  the  whole  gelatine  liquefied. 

Staining.  —Do  not  take  aniline  dyes  very  well.  Gram's  method 
is,  however,  applicable. 

Pathogenesis. — If  a  pure  culture  is  spread  over  the  honey- 
comb containing  bee  larvae,  or  if  bees  are  fed  upon  infected 
material,  foul-brood  disease  will  occur.  Mice,  if  injected,  die  in  a 
few  hours.  (Edema  around  the  point  of  infection,  and  many 
bacilli  contained  in  the  cedematous  fluid,  otherwise  no  changes. 

Micrococcus  Amylovorus    (Burrill.) 

Origin.—  In  the  disease  called  "Blight,"  which  affects  pear- 
trees  and  other  plants. 

Form.— Small  oval  cells,  never  in  chains,  more  the  form  of  a 
bacillus. 

Pathogenesis. — Introduced  into  small  incisions  in  the  bark  of 
pear-trees  the  trees  perished  from  the  "  blight."  The  starch  of 
the  plant  cell  was  converted  into  carbon  dioxide,  hydrogen, 
and  butyric  acid. 

Bacterium  Termo.    (Cohn.) 

This  was  a  name  given  to  a  form  of  micro-organism  found  in 
decomposing  albuminous  material,  and  was  supposed  to  be  one 
specific  germ.  Hauser,  in  1885,  found  three  different  distinct 
microbes  which  he  grouped  under  the  common  name  of  Proteus, 
which  have  the  putrefying  properties  ascribed  to  B.  Termo. 

Proteus  Vulgaris. 

Origin. — In  putrid  animal  matter,  in  the  feces,  and  in  water. 

Form. — Small  rods,  slightly  curved,  of  varying  lengths,  often 
in  twisted  chains,  having  long  cilia  or  flagella. 

Properties.—  Very  motile,  and  very  soon  liquefying  gelatine  ; 
forms  hydrogen  sulphide  gas  ;  causes  putrefaction  in  meat. 

Growth. —Growth  very  rapid,  best  at  24°  C,  is  facultative 
aerobic. 

Gelatine  Plates. — Yellowish-brown,  irregular  colonies,  with 
prolongations  in  every  direction,  forming  all  sorts  of  figures  ;  an 


BACTERIA    PATHOGENIC    FOR    ANIMALS.  171 

impression  preparation  shows  these  spider-leg  processes  to  con- 
sist of  bacilli  in  regular  order. 

Stab  Culture. — The  gelatine  soon  liquid,  a  gray  layer  on  the 
surface,  but  the  chief  part  of  the  culture  in  small  crumbs  at  the 
bottom. 

Pathogenesis.— Rabbits  and  guinea-pigs  injected  subcutane- 
ously  die  quickly,  a  form  of  toxaemia,  hemorrhagic  condition  of 
lungs  and  intestines  present.  When  neurin  is  injected  previ- 
ously the  animals  do  not  die.  This  ptomaine  is  supposed  to  be 
generated  by  the  proteus  vulgaris. 

Proteus  Mirabilis.     (Hauser.) 

Differs  from  P.  vulgaris  in  that  the  gelatine  is  less  rapidly 
liquefied.     Found  also  in  putrid  material. 

Proteus  Zenkeri.     (Hauser.) 

Does  not  liquefy  gelatine  ;  otherwise  similar  to  the  other  two. 

We  have  now  considered  some  of  the  characteristics  of  the 
more  important  bacteria.  The  scope  of  this  work  does  not  allow 
a  more  extended  study  than  we  have  made,  which,  as  we  are 
aware,  has  been  very  superficial.  The  larger  works  must  be 
referred  to,  if  a  deeper  interest  is  taken  in  the  subject. 


APPENDIX 


YEASTS  AND  MOULDS. 

In  works  on  bacteria,  these  true  fungi,  yeasts  and  moulds,  are 
usually  considered.  They  are  so  closely  related  to  bacteria,  and 
so  often  contaminate  the  culture  media,  and  are  so  similar  in 
many  respects,  that  a  description  is  almost  a  necessity. 

But  there  are  several  thousand  varieties,  and  we  cannot 
attempt  to  describe  even  all  of  the  more  important  ones.  It 
will  answer  our  purpose  to  detail  a  few  of  the  more  common 
kinds,  and  give  the  principal  features  of  the  different  orders. 

' Saccharomycetes  or  Yeasts  increase  through  budding;  the 
spores  are  attached  to  the  mother  cell  like  a  tuber  on  a  potato. 

Yeasts  are  the  cause  of  alcoholic  fermentation  in  the  saccha- 
roses.    A  description  of  the  most  common  ones  will  suffice. 

Saccharomyces  Cerevisise.  (Torula  Cerevisice.)  This  is  the 
ordinary  beer  yeast. 

Form. — Bound  and  oval  cells ;  a  thin  membrane  inclosing  a 
granular  mass,  in  which  usually  can  be  seen  three  or  four  irre- 
gular-shaped spores.  When  these  become  full  grown  they  pass 
through  the  cell  wall  and  form  a  daughter  cell.  Sometimes  long 
chains  are  produced  by  the  attached  daughter  cells. 

Growth.—  They  can  be  cultivated  as  bacteria  in  bouillon,  but 
they  grow  best  in  beer. 

There  are  several  varieties  of  beer  yeast,  each  one  giving  a 
characteristic  taste  to  the  beer.  Brewers,  by  paying  special 
attention  to  the  nutrient  media,  cultivate  yeasts  which  give  to 
their  beers  individual  flavors. 

Mixed  yeast  gives  rise  to  a  poor  quality  of  beer. 

Saccharomyces  Rosaceus.  S.  Niger  and  S.  Albicans.  These 
yeasts  are  found  in  the  air ;  and  instead  of  producing  alcoholic 

(173) 


174  APPENDIX. 

fermentation  they  give  rise  to  a  pigment  in  the  culture  media. 
They  grow  upon  gelatine  which  they  do  not  liquefy. 

Saccharomyces  Mycoderma.  This  yeast  forms  a  mould-like 
growth,  a  skin,  on  the  surface  of  fermented  liquids,  but  does  not 
cause  any  fermentation  itself.  It  forms  the  common  u  mould" 
on  wine,  preserves,  and  "  sour  krout." 

Pathogenic  Yeasts.  In  recent  years  a  number  of  workers 
have  interested  themselves  in  experiments  with  yeasts  in  their 
relation  to  disease;  and  under  the  name  of  Blastomycetes,  San- 
felice  has  grouped  yeasts  that  produce  tumors  resembling  epi- 
theliomata ;  and  he  has  tried  to  prove  that  the  so-called  animal 
parasites  found  in  malignant  growths,  and  variously  known  as 
coccidia  and  sporozoa,  are  yeasts.  The  whole  subject  is  still 
under  discussion. 

Oidium.  A  form  which  seems  to  be  the  bridge  between  the 
yeast  and  the  moulds  is  the  oidium.  Sometimes  it  resembles 
the  yeasts,  sometimes  the  moulds,  and  often  both  forms  are 
found  in  the  same  culture.     Several  are  pathogenic  for  man. 

Oidium  Lactis. 

Origin. — In  sour  milk  and  butter. 

Form.—  The  branches  or  hyphens  break  up  into  short  rod-like 
spores.     No  sporangium,  as  in  moulds. 

Growth.— In  milk  it  appears  as  a  white  mould. 

Artificially  cultured  on  gelatine  plates,  or  milk  gelatine  plates, 
it  forms  satin-like,  star-shaped  colonies,  which  slowly  liquefy. 
Under  microscope  the  form  of  the  fungus  is  well  seen. 

Agar  Stroke  Culture.— The  little  stars,  very  nicely  seen  at  first ; 
then  the  culture  becomes  covered  with  them,  causing  a  smeared 
layer  to  appear  over  the  whole  surface,  with  a  sour  odor. 

Properties. — The  milk  is  not  changed  in  any  special  way.  It 
is  not  pathogenic  for  man  or  animals.  It  is  found  when  the 
milk  begins  to  sour. 

Oidium  Albicans.    {Soor.)    Thrush  Fungus. 

Origin. — Mucous  membrane  of  the  mouth,  especially  of  infants. 

Form. — Taken  from  the  surface  of  the  culture,  a  form  like 
yeasts  ;  but  in  the  deeper  layers,  mycelia  with  hyphens  occur. 

Growth. — Not  liquefying;  snow-white  colonies  on  gelatine 
plates. 


YEASTS    AND    MOULDS.  17o 

Stab  Culture.—  Radiating  yellow  or  white  processes  spring  from 
the  line  made  by  the  needle,  those  near  the  surface  having  oval 
ends. 

Potatoes.—  The  yeast  form,  develops  as  thick  white  colonies. 

Bread  Mash. — Snow-white  veil  over  the  surface. 

Pathogenesis. — In  man  the  parasitic  thrush,  or  "white  mouth, n 
is  caused  by  this  fungus.  In  the  white  patches  the  spores  and 
filaments  of  this  microbe  can  be  found.  Rabbits  receiving  an 
intravenous  injection  perish  in  twenty-four  to  forty-eight  hours, 
the  viscera  being  filled  with  mycelia. 

True  Moulds.  Fliigge  has  made  five  distinct  divisions  of 
moulds.  It  will,  however,  serve  our  purpose  to  classify  those 
to  be  described  under  three  headings :  Penicillium,  Mucor,  and 
Aspergillus. 

Penicillium  Glaucum. 

Origin.— The  most  widely  distributed  of  all  moulds,  found 
wherever  moulds  can  exist. 

Form. — From  the  mycelium,  hypha?  spring  which  divide  into 
basidia  (branches),  from  which  tiny  filaments  arise  (sterigmata ), 
arranged  like  a  brush  or  tuft.  On  each  sterigma  a  little  bead 
or  conidium  forms,  which  is  the  spore.  In  this  particular  fungus 
the  spores  in  mass  appear  green. 

Growth. — It  develops  only  at  ordinary  temperatures,  forming 
thick  grayish-green  moulds  on  bread-mash.  At  first  these  ap- 
pear white,  but  as  soon  as  the  spores  form,  the  green  predomi- 
nates.    Gelatine  is  liquefied  by  it. 

Mucor  Mucedo.  Next  to  the  penicillium  glaucum,  this  is  the 
most  common  mould.  Found  in  horse  dung,  in  nuts,  and 
apples,  in  bread  and  potatoes  as  a  white  mould. 

Form. — The  mycelium  sends  out  several  branches,  on  one  of 
which  a  pointed  stem  is  formed  which  enlarges  to  form  a  globu- 
'ar  head,  a  spore-bulb,  or  Sporangium.  The  spore-bulb  is  par- 
ilioned  off  into  cells  in  which  large  oval  spores  lie.  When  the 
spores  are  ripe  a  cap  forms  around  the  bulb,  the  walls  break 
down  and  the  wind  scatters  the  spores,  leaving  the  cap  or 
M  columella"11  behind. 

Growth. — Takes  place  at  higher  temperatures  on  acid  media. 

It  is  not  Pathogenic. 


176 


APPENDIX 


Achorion  Schbnleinii. 

Trichophyton  Tonsurans. 

Microsporon  Furfur. 

These  three  forms  are  similar  to  each  other  in  nearly  every 
particular  and  resemble  in  some  respects  the  oidium  lactis,  in 
other  ways  the  mucors.  The  first  one,  Achorion  Schbnleinii,  was 
discovered  by  Schonlein  in  1839,  in  Favus,  and  is  now  known  as 
the  direct  cause  of  this  skin  disease. 

Fig.  91. 


Achorion  Schbnleinii  (after  Kaposi). 


Origin, — Found  in  the  scaly  crusts  of  favus. 

Form. — Similar  to  oidium  lactis. 

Growth. — Is  very  sparse.  On  gelatine  round  white  masses 
inclosed  by  a  zone  of  liquefied  gelatine. 

In  milk  it  is  destroyed. 

Pathogenesis. — Causes  favus  in  man. 

Trichophyton  Tonsurans.    Found,  in  1854,  by  Bazin,  in  Tinea. 

Form. — Similar  to  the  achorion  or  favus  fungus. 

Growth. — Somewhat  more  rapid  than  the  favus,  and  the  gela- 
tine quickly  liquefied,  Old  cultures  are  of  an  orange-yellow 
color.     Colonies  have  a  star-shaped  form. 

Pathogenesis.  —  Herpes  tonsurans  and  the  various  tineee  are 
produced  by  this  fungus. 

Microsporon  Furfur.    Found  in  tinea  versicolor,  almost  iden- 


YEASTS    AND    MOULDS.  177 

tical  with  the  above,  forms  dry  yellow  spots,  usually  on  the  chest 
in  persons  suffering  from  wasting  diseases. 

Aspergillus  Glaucus. 

Origin. — In  saccharine  fruits. 

Form. — The  hypha  has  formed  upon  its  further  end  a  bulb, 
from  which  pear-shaped  sterigmata  arise  and  bear  upon  their 
ends  the  conidia  or  spores. 

Growth.— Best  upon  fruit  juices.  Non-pathogen  fc.  The  mould 
is  green.  Aspergillus  flams  has  the  tufts  and  spores  of  a  yellow 
color. 

A.  Fumigatus.  Is  pathogenic  for  rabbits  when  injected  into 
them.  At  the  autopsy  their  viscera  are  found  filled  with  the 
mould. 

Examination  of  Yeasts  and  Moulds.  Yeasts  and  moulds  are 
best  examined  in  the  unstained  condition.  A  small  portion  of 
the  colony  rubbed  up  with  a  mixture  of  alcohol  and  a  few  drops 
of  liquor  ammonia  ;  of  this,  a  little  is  brought  upon  the  glass- 
slide  covered  with  a  drop  of  glycerine  and  the  cover-glass  pressed 
upon  it.  If  the  preparation  is  to  be  saved,  the  cover-glass  is 
secured  by  ringing  around  the  edges.  Yeasts  take  methylin-blue 
stain  very  well. 

Cladothrices  and  Streptothrices.  The  streptothrix  and 
cladothrix  groups  are  classed  with  the  higher  bacteria,  but 
their  exact  status  is  still  undetermined.  They  may  be  consid- 
ered as  representing  the  transition  from  the  bacteria  to  the 
lower  fungi. 

Streptothrix,  or  Cladothrix  Actinomyces  (ray  fungus). 
Actinomycosis  is  a  disease  caused  in  man  and  cattle  by  this 
organism,  which  is  commonly  found  in  grain,  particularly 
barley.  It  is  probable  that  several  varieties  of  the  parasite  can 
produce  the  characteristic  lesions.  It  has  been  discovered  in 
all  countries  and  in  various  organs  of  the  body,  although  its 
place  of  election  is  about  the  lower  jaw,  where  it  tends  to  form 
hard  ulcerating  abscesses,  affecting  other  organs  secondarily. 

Form. — In  the  granular  masses  of  an  abscess  cylindrical  fila- 
ments are  matted  together,  and  radiating  outward  from  this 
zone  are  club-shaped  branches,  as  the  petals  of  an  aster.  In  the 
center  of  the  granule  are  numerous  cocci-like  bodies,  and  some 

12 


178  APPENDIX. 

of  the  ovoid  or  club-shaped  hyphae  lie  detached  from  the 
clusters.  Through  cultivation  it  was  found  that  the  ovules  give 
rise  to  filaments,  and  they  then  form  the  ovules  again. 

Cultivation. — At  38°  C.  on  glycerine-agar  in  a  period  of  one 
to  two  weeks,  pointed  scales  about  the  size  of  a  millet-seed, 
center  dry  and  prominent,  margins  hyaline,  composed  only  of 
filaments,  short  and  long,  massed  together,  but  no  clubbed 
forms. 

By  some  the  clubs  are  considered  the  spore  organs ;  by  others 
they  are  thought  to  be  encapsulated  or  thickened  filaments. 

Pathogenesis. — When  a  portion  of  the  growth  obtained  in 
eggs  was  injected  into  the  abdominal  cavity  of  a  rabbit,  actinomy- 
cotic processes  developed  upon  the  peritoneum. 

It  usually  gains  access  to  the  living  body  through  a  wound  in 
the  gum  or  some  caries  of  the  teeth.  A  new  growth  is  formed, 
ulceration  being  first  set  up. 

The  new  tissue,  composed  of  round  cells,  then  undergoes  soft- 
ening, purulent  collections  form  and  the  normal  structure  is 
destroyed. 

The  usual  seat  is  in  the  maxillary  bones,  but  the  fungus  has 
been  found  in  the  lungs,  tonsils,  intestines,  and  various  other 
organs  in  man  and  cattle. 

Examination. — Well  seen  in  the  unstained  condition.  From 
the  pus  or  scraping  a  small  portion  is  taken  and  squeezed  upon 
the  glass  slide  ;  if  calcareous  matter  is  present,  a  drop  of  nitric 
acid  will  dissolve  the  same. 

Glycerine  will  preserve  the  preparation. 

Staining.—  Cover-glass  specimens  stained  best  with  Gram's 
method.     Tissue  sections  should  be  stained  as  follows  :  — 

Ziehl's  carbol-fuchsin,  ten  minutes.     Kinse  in  water. 

Cone,  alcohol. sol.  of  picric  acid,  five  minutes.     Rinse  in  water. 

Alcohol,  50  per  cent.,  fifteen  minutes.  Alcohol  absolute,  clove 
oil,  balsam. 

The  rays  stained  red,  the  tissue  yellow. 

Streptothrix  Madurae.    (Vincent.) 

Origin. — Found  in  the  disease  known  as  Madura  foot,  or 
Mycetoma,  an  ulceration  affecting  the  feet,  especially  of  indi- 


YEASTS    AND    MOULDS 


179 


viduals  living  in  the  tropics.     Two  varieties,  the  pale  and  the 
black,  have  been  described. 

Form. — Branched  filaments  resembling  the  actinomyces  strep- 
tothrix  in  the  mycelia.     Spores  are  seen. 

Cultivation. — In  liquid  media  containing  vegetable  infusions 
growth  occurs  best.     Temper- 
ature of  37°  C.  most  suited.  Fig.  92. 
The  colonies  near  the  surface 
become  colored  red. 

Agar. — Glazed  colonies,  at 
first  colorless,  then  rose-col- 
ored, about  the  size  of  a  pea, 
with  the  central  part  umbili- 
cated  and  pale.  Gradually  the 
rose  color  fades. 

Acid  Potato. — A  slow  and 
meager  growth. 

Pathogenesis. — Only  local  re- 
action has  been  caused  by  in- 
oculation in  animals.  In  man 
the  disease  usually  follows  a 
slight  injury  and  attacks  the  leg 
or  foot,  slowly  forming  a  nodu- 
lar growth,  which  in  the  course 
of  months  or  a  year  begins  to 
soften  and  ulcerate,  and  with 
the  sero-pus  are  discharged 
numerous  little  granules,  some 
black,  some  pink,  containing 
mycelia.  The  limb  becomes 
much    deformed,    the    tissue 


■WP< 


• 


M 


Streptothrix  Madura;  in  a  secti 

eased  tissue(Vincent) 


dis- 


vascularized,  and  the  degenerated  area. filled  with  the  strep- 
tothrix filaments.^ 

Staining. — The  organism  itself  stained  with  ordinary  stains. 
Gram's  method  for  the  tissue. 

Streptothrix  Farcinica.  (Nocard.)  Bovine  Farcy,  Farcin  du 
Boeuf. 


180  APPENDIX. 

Origin. — A  disease  affecting  cattle  and  giving  rise  to  tubercle- 
like lesions  in  the  lungs,  liver,  and  spleen.    Common  in  France. 

Form. — Small  interwoven  mass  of  threads  arranged  in  tufts 
found  in  the  centers  of  the  tubercles. 

Culture. — At  body-temperature  in  various  media. 

Bouillon. — Colorless  masses  irregular  in  size  and  shape. 

Agar  and  Gelatine. — Small,  rounded,  opaque  colonies,  thicker 
at  the  periphery. 

Potato. — Rapid  growth  of  pale  yellow  dry  scales,  consisting  of 
many  spores. 

Pathogenesis. — Pure  cultures  introduced  into  the  peritoneum 
of  guinea-pigs  give  rise  in  9  to  20  days  to  tubercle-like  lesions. 
Subcutaneous  injections  cause  abscesses  with  secondary  in- 
volvement of  the  lymphatics,  ending  in  recovery.  Dogs, 
horses,  and  rabbits  are  immune. 

Staining. — Wright's  double  stain  for  tissues;  also  Gram's. 

Examination  of  Air,  Soil,  and  Water. 

Air. — Many  germs  are  constantly  found  in  the  atmosphere 
about  us.  Bacteria  unaided  do  not  rise  into  the  air  and  fly 
about ;  they  usually  become  mixed  with  small  particles  of  dirt 
or  dust  and  are  moved  with  the  wind.  The  more  dust  the  more 
bacteria,  and  therefore  the  air  in  summer  contains  a  greater 
number  than  the  air  in  winter,  and  all  the  other  differences  can 
be  attributed  to  the  greater  or  less  quantity  of  dust  and  wind. 

Methods  of  Examination.  The  simplest  method  is  to  ex- 
pose a  glass  or  dish  covered  with  gelatine  in  a  dust-laden 
atmosphere  or  in  the  place  to  be  examined.  In  the  course  of 
24  to  48  hours  colonies  will  be  seen  formed  wherever  a  germ  has 
fallen.  But  this  method  will  not  give  any  accurate  results  in 
regard  to  the  number  of  bacteria  in  a  given  space  ;  for  such  a 
purpose  somewhat  more  complicated  methods  are  needed,  so 
that  a  certain  amount  of  air  can  come  in  contact  with  the 
culture  media  at  a  certain  regulated  rate  of  speed. 

Hesse's  Method.  This  is  the  most  useful  of  the  various 
methods  in  vogue. 

A  glass  cylinder,  70  centimetres  long  and  3.5  centimetres  in 
diameter,  is  covered  at  one  end,  by  two  rubber  caps,  the  inner 


AIR,    SOIL,    AND    WATER. 


181 


one  having  a  hole  in  its  centre  10  millimetres  in  diameter  ;  and 
at  the  end  B  a  rubber  cork  fits  in  the  cylinder;  through  this 
cork  a  glass  tube  10  mm.  in  diameter  passes,  which  is  plugged 
at  both  ends  with  cotton.  The  cylinder  and  fittings  are  first 
washed  in  alcohol  and  sublimate  and  then  placed  for  one  hour 
in  the  steam  chamber. 

Removing  the  cork  of  the  cylinder,  50  cubic  centimetres  of 
sterile  gelatine  in  a  fluid  condition  are  introduced  and  rolled 
out  on  the  sides  of  the  tube,  after  the  manner  of  Esmarch, 
leaving  a  somewhat  thicker  coating  along  the  under  side  of  the 

Fig.  93. 


cylinder.  The  aeroscope,  as  the  cylinder  and  its  fittings  are 
called,  is  placed  upon  an  ordinary  photographer's  tripod  and 
the  glass  tube,  which  passes  through  the  rubber  cork,  connected 
with  an  aspirator,  the  cotton  having  first  been  removed  from  its 


82 


APPENDIX. 


Fig.  94. 


outer  end.  The  aspirator  consists  of  two  ordinary  wash-bottles 
connected  with  each  other  by  a  rubber  tube,  0.  They  are  at- 
tached to  the  tripod  with  a  small  hook  one  above  the  other,  the 
upper  one  half  filled  with  water  and  slightly  tilted. 

When  the  apparatus  is  wanted,  the  outer  rubber 
cap  at  the  end  A  of  the  aeroscope  is  removed,  the 
air  can  then  pass  through  the  small  hole  in  the 
other  cap,  and  the  germs  fall  upon  the  gelatine  in 
the  tube,  the  cotton  in  the  small  glass  tube  at  the 
other  end  preventing  the  germs  from  getting  out. 
The  aspirator  is  set  in  use  by  tilting  the  upper 
bottle  so  that  the  water  flows  into  the  lower,  this 
creates  suction  and  draws  the  air  through  the 
aeroscope. 

The  amount  entering  estimated  by  the  capacity 
of  the  wash-bottle.  The  rate  at  which  it  enters 
depending  upon  the  rate  of  the  flow  of  water, 
which  can  be  regulated. 

Hesse  advises  for  rooms  and  closed  spaces  1  to  5 
litres,  at  the  rate  of  2  minutes  a  litre,  and  for  open 
spaces,  10  to  20  litres  at  4  minutes  a  litre.  Plate 
cultures  can  be  made  from  the  colonies  which  de- 
velop in  8  to  10  days  in  the  cylinder. 

Petri's  Method.  The  air  pumped  or  sucked 
through  sand  filters,  and  the  sand  then  mixed  with 
gelatine. 

Sand  is   sterilized   by  heating  to  redness,  and 
while  still  warm  placed  in  test  tubes  which  are 
Sand  filter        then   plugged.      (Sand   which    has    been    passed 
a  ter   e  n.        through  a  sieve  with  meshes  0.25  millimetre  wide 
is  the  kind  required.)     A  glass  tube  9  centimetres  long  is  pro- 
vided with  two  portions  of  sand  each  3  cm.  long  and  £  cm.  apart, 
little  plates  of  brass  gauze  keeping  the  portions  in  position. 

The  tube  and  its  contents  now  sterilized  in  hot  air  oven  at 
150°  C,  the  ends  having  first  been  plugged  with  cotton. 

One  end  of  the  tube  is  then  fitted  with  a  rubber  cork  through 
which  passes  a  glass  tube,  which  is  connected  with  an  aspirator 
(a  hand-pump  with  a  known  capacity). 


AIR,   SOIL,    AND    WATER. 


183 


If  a  hundred  litres  of  air  pass  through  the  tube  in  fifteen  min- 
utes the  germs  should  all  be  arrested  in  the  first  sand  filter. 

And  when  the  filters  are  removed  and  thoroughly  mixed  with 
gelatine,  each  filter  for  itself,  there  should  be  no  colonies  de- 
veloped from  the  second  filter,  i.  c,  the  one  nearest  the  aspirator. 

Sedgwick-Tucker  Method.  A  special  form  of  tube  is  used, 
called  an  aerobioscope.    It  consists  of  a  neck  2.5  cm.  in  length, 


Fig.  95. 


Sedgwick-Tucker  aerobioscope. 


an  expanded  portion  15  cm.  long,  and  a  long  narrow  tube  of  15 
cm.  After  sterilization  the  tube  is  partly  filled  with  granulated 
sugar,  which  is  the  filtering  material.  By  means  of  a  vacuum 
gauge  and  an  air-pump,  or  ordinary  aspirating  bottles,  the  vol- 
ume of  air  passing  through  the  apparatus  can  be  determined. 
After  the  air  has  been  passed  through,  the  sugar  is  gently 
shaken  from  the  narrow  tube  into  the  expanded  portion,  and 
20  c.  c.  of  liquefied  gelatine  is  poured  in.  The  sugar  dissolves, 
and  the  mixture  is  then  rolled  on  the  inner  side  of  the  glass 
as  an  Esmarch  tube.  This  part  of  the  apparatus  is  divided  into 
squares  to  make  the  counting  of  colonies  easy.  The  aerobio- 
scope is  very  highly  recommended. 

Varieties  Found  in  Air.  The  only  pathogenic  bacteria  found 
with  any  constancy  are  the  staphylococcus  aureus  and  citreus; 
but  any  bacterium  can  be,  through  accident,  lifted  into  the  atmo- 
sphere, and  in  certain  places  may  be  always  found — the  bacillus 
tuberculosis,  for  example,  in  rooms  where  many  consumptives 
are  living. 

Non-Pathogenic.  The  micrococci  predominate.  Sarcinse, 
yeasts,  and  moulds  constantly  contaminate  cultures. 

In  the  ordinary  habitations  the  average  number  of  germs  to 
the  litre  of  air  does  not  exceed  five. 

Around  water-closets,  where  one  would  imagine  a  great  num- 
ber to  exist,  owing  to  the  undisturbed  condition  of  the  air,  but 
few  will  be  found. 


184  APPENDIX. 

Examination  of  Water.  The  bacteriological  examination  of 
water  is  to-day  of  as  much  importance  as  the  chemical  analy- 
sis, and  must  go  hand  in  hand  with  it. 

At  the  start  we  must  say  that  a  water  containing  thousands 
of  germs  to  the  cubic  centimeter  is  far  less  dangerous  than  one 
containing  but  two  germs,  if  one  of  these  two  be  a  typhoid  ba- 
cillus. It  is  not  the  number  that  proves  dangerous,  it  is  the 
kind. 

If  a  natural  water  contains  more  than  500  germs  to  the  cubic 
centimeter,  it  were  well  to  examine  its  source. 

Bacteriology  performs  the  greatest  service  in  testing  the  devices 
which  are  intended  to  render  water  fit  for  drinking. 

As  a  diagnostic  aid  the  examination  is  of  but  little  use.  An 
epidemic  of  typhoid  fever  occurs,  the  water  is  suspected,  an  ex- 
amination is  undertaken  ;  but  the  days  of  incubation  and  the 
days  passed  before  the  water  is  analyzed  have  given  the  typhoid 
germs,  if  any  had  been  present,  ample  time  to  disappear,  since 
in  water  that  contains  other  bacteria  they  live  a  very  short  time 
only.  Again,  the  water  tested  one  day  may  be  entirely  free  and 
the  next  day  contain  a  great  number,  and  before  the  typhoid 
germ  can  be  proven  to  be  present  in  that  particular  water,  the 
epidemic  may  be  past. 

Purity  of  Waters.  The  purest  water  we  have  is  the  natural 
spring  water— water  that  has  slowly  filtered  its  way  through 
various  layers  of  gravel  and  sand  and  comes  finally  clear  and 
sparkling  from  the  ground.  It  is  without  germs  ;  but  let  such 
a  water  stand  walled  up  in  cisterns  or  wells,  it  becomes  as 
surface  water,  open  to  all  sorts  of  impurities,  and  the  bacterial 
nature  of  it  changes  every  moment. 

Artesian  or  Driven  Well.  The  driven  well  will  secure  to  a  cer- 
tain extent  a  pure  water.  It  is  the  only  form  of  well  or  cistern 
that  will  insure  this,  since  the  water  does  not  become  stagnant 
in  it ;  but  it  may  connect  with  an  outhouse,  the  soil  being  very 
loose,  allowing  the  products  of  germs  of  refuse  water  to  find  their 
way  into  the  well.  If  a  chemical  examination  shows  increased 
amounts  of  chloride  of  sodium,  a  contamination  can  be  mooted. 

Filtered  Water.  Dangerous  as  surface  water  is,  the  greater 
quantity  used,  is  such  :  the  inhabitants  of  larger  towns  and  cities 


AIR,     SOIL,     AND    WATER.  185 

using  chiefly  the  rivers  and  other  large  waters  which  course 
near  them  for  drinking  purposes.  A  purification  or  filtration 
can  in  a  certain  measure  render  these  waters  harmless. 

Filtration  is  often  carried  on  on  a  large  scale  in  the  water- 
works of  cities  and  towns. 

Bacteriological  examination  is  here  of  great  service  to  deter- 
mine if  a  water,  which  has  been  filtered  and  may  have  a  very 
clear  appearance,  and  give  no  harmful  chemical  reaction,  yet 
be  entirely  free,  or  nearly  so,  from  germs ;  in  other  words,  if 
the  filter  is  a  germ  filter  or  not. 

Charcoal  Sponge  and  Asbestos,  the  materials  formerly  in  use, 
are  objectionable  because  germs  readily  develop  on  them  and 
clog  them,  so  that  they  require  frequent  renewal.  In  very 
large  filters,  sand  and  gravel  give  the  best  results ;  the  number 
of  germs  in  a  cubic  centimetre  is  reduced  to  forty  or  fifty  and 
kept  at  that  number.  This  is  a  very  pure  water  for  a  city  water, 
though,  as  we  stated  before,  not  a  safe  one,  for  among  those 
forty  germs  very  dangerous  ones  may  be  found.  It  is  then 
necessary  for  the  users  to  refilter  the  water  before  drinking  it, 
through  a  material  which  will  not  allow  any  germs  to  pass. 

Pasteur-Chamberland  Filter.  This  very  perfect  filter,  which 
is  now  in  almost  universal  use,  consists  of  a  piece  of  polished 
porcelain  in  the  form  of  a  cylinder  closed  at  one  end  and  pointed 
at  the  other.  It  is  placed  in  another  cylinder  of  glass  or  rubber 
and  the  pointed  portion  connected  with  a  bottle  containing  the 
water,  or  directly  with  faucet  of  the  water-pipe.  The  water 
courses  through  the  porcelain  very  slowly  and  comes  out  entirely 
free  from  germs ;  pipe-clay,  bisque,  infusorial  earth,  and  kaolin 
are  also  perfect  filters.  The  only  disadvantage  is  the  long  time 
it  takes  for  the  water  to  pass  through.  Pressure  is  used  to 
accelerate  the  passage  in  the  form  of  an  aspirator  or  air-pump. 

The  force  of  the  hydrant  water  is  also  sufficient  to  produce  a 
steady,  small  stream. 

These  porcelain  cylinders  can  easily  be  sterilized  and  the 
pores  washed  out. 

All  the  cylinders  or  bougies  are  not  germ  proof,  so  that  they 
must  be  tested,  and  most  of  them  must  be  cleaned  every  fourth 
day,  or  they  will  allow  germs  to  pass  through. 


186  APPENDIX. 

Boiling  as  a  means  of  purifying.  When  such  a  filter  cannot 
be  obtained,  the  only  alternative  is  to  boil  all  the  water  to  be 
used  for  drinking ;  and  this  should  especially  be  done  in  times 
of  typhoid  and  cholera  epidemics. 

Methods  of  Examination.  Since  the  germs  rapidly  multiply 
in  stagnant  water,  an  examination  must  not  be  delayed  longer 
than  an  hour  after  the  water  has  been  collected.  Every  pre- 
caution must  be  taken  in  the  way  of  cleanliness  to  prevent  con- 
tamination ;  sterilized  flasks,  pipettes,  and  plugs  must  be  at 
hand,  and  the  gelatine  tubes  best  inoculated  on  the  spot.  If 
this  cannot  be  done,  the  sample  should  be  packed  in  ice  until 
it  arrives  at  the  laboratory.  The  sample  is  placed  in  a  steri- 
lized glass  flask,  and  the  flask  then  closed  with  a  sterile  cotton 
plug.  A  sterilized  pipette  is  then  dipped  into  the  flask  and 
1  c.c.  of  the  water  withdrawn  and  added  to  a  tube  of  gelatine, 
the  gelatine  being  in  a  fluid  condition.  To  a  second  tube, 
£  c.c.  is  added.  The  tubes  are  then  shaken  so  as  to  thor- 
oughly mix  the  water  with  the  gelatine,  and  then  poured 
upon  wide  glass  plates,  one  plate  for  each  tube ;  the  plates  are 
then  placed  in  the  moist  chamber,  and  in  two  or  three  days 
examined.  A  temperature  of  18°  to  20°  C.  is  best.  Many  water- 
bacteria  are  hindered  by  higher  degrees  of  heat.  If  the  germs 
are  equally  divided,  there  should  be  one-half  the  number  on 
one  plate  that  there  is  on  the  other;  thus  the  ?  c.c.  serves  as 
control. 

Water  that  is  very  rich  in  germs  requires  dilution  with  ster- 
ilized water  50  to  100  times.  Fewer  colonies  will  be  found  on 
agar  than  on  gelatine  even  at  the  same  temperature. 

To  count  the  colonies  which  develop  upon  the  plates,  a  spe- 
cial apparatus  has  been  designed,  known  as 

Wolfhiigel's  Apparatus.  A  glass  plate  divided  into  squares, 
each  a  centimeter  large,  and  some  of  these  subdivided.  This 
plate  is  placed  above  the  gelatine  plate  with  the  colonies,  and 
the  number  in  several  quadrants  taken,  a  lens  being  used  to 
see  the  smaller  ones. 

The  petri  saucers  can  be  used  instead  of  plates,  and  an  appa- 
ratus on  the  Wolf  hiigel  plan  can  be  obtained  to  count  the  colo- 
nies.   It  is  best  to  count  all  the  colonies  on  the  plate  or  dish. 


AIR,     SOIL,     AND     WATER.  187 

Agar  and  bouillon  are  used  in  qualitative  analyses.  A  large 
quantity  of  the  water  is  taken  (about  100  c.  cm.)  and  mixed 
with  25  c.c.  of  bouillon ;  the  mixture  is  then  placed  in  an  in- 
cubator. The  ordinary  water-bacteria  do  not  bear  the  higher 
temperatures  very  well,  and  therefore  pathogenic  organisms— 
as  cholera,  for  instance — will  be  found  almost  in  pure  cultures. 

The  growth  of  intestinal  bacteria  is  also  favored  by  glucose 
bouillon  (2  per  cent.),  and  fermentation  ensues.  If  a  fermenta 
tion-tube  (Smith's)  is  used,  the  gas  collects  at  one  end,  and  the 
bacteria  can  be  further  cultivated  and  studied. 

Varieties  Found.  The  usual  kinds  found  are  non-patho- 
genic, but,  as  is  well  known,  typhoid  and  cholera  are  princi- 
pally spread  through  drinking  water,  and  many  other  germs 
may  find  their  way  into  the  water.  Some  of  the  common 
varieties  give  rise  to  fluorescence,  or  produce  pigment. 

Eisenberg  gives  100  different  varieties  as  ordinarily  found. 
Other  intestinal  diseases  also  are  supposed  to  be  water  borne,  and 
the  presence  of  large  numbers  of  the  Bacillus  coli  communis  is 
strongly  suggestive  of  sewage  contamination.  Ice  supplies 
require  the  same  supervision  as  water  supplies,  for  many  bac- 
teria, like  the  typhoid  bacillus,  retain  their  vitality  for  weeks 
after  freezing. 

The  Examination  of  the  Soil.  The  upper  layers  of  the  soil 
contain  a  great  many  bacteria,  but  because  of  the  difficulty  in 
analyzing  the  same,  the  results  are  neither  accurate  nor  con- 
stant. The  principal  trouble  lies  in  the  mixing  of  the  earth 
with  the  nutrient  medium  ;  little  particles  of  ground  will  cling 
to  the  walls  of  the  tube,  or  be  imbedded  in  the  gelatine,  and 
may  contain  within  them  myriads  of  bacteria.  As  with  water, 
the  soil  must  be  examined  immediately  or  very  soon  after  it  is 
collected,  the  bacteria  rapidly  multiplying  in  it. 

When  the  deeper  layers  are  to  be  examined,  some  precautions 
must  be  taken  to  avoid  contamination  with  the  other  portions  of 
the  soil.  One  method,  very  laborious  and  not  often  practical,  is 
to  dig  a  hole  near  the  spot  to  be  examined  and  take  the  earth 
from  the  sides  of  this  excavation. 

Frankel's  Borer.  Frankel  has  devised  a  small  apparatus  in 
the  form  of  a  borer,  which  contains  near  its  lower  end  a  small 


188  APPENDIX. 

cavity,  which  can  be  closed  up  by  turning  the  handle,  or  opened 
by  turning  in  the  opposite  direction. 

It  is  introduced  with  the  cavity  closed,  and  when  it  is  at  the 
desired  depth,  the  handle  is  turned,  the  earth  enters  the  cavity, 
the  handle  again  turned,  incloses  it  completely,  and  the  borer  is 
then  withdrawn. 

The  earth  can  then  be  mixed  with  the  gelatine  in  a  tube,  and 
this  gelatine  then  rolled  on  the  walls  of  the  tube  after  the  man- 
ner of  Esmarch,  or  it  can  be  poured  upon  a  glass  plate,  and  the 
colonies  developed  so. 

Another  method  is  to  wash  the  earth  with  sterilized  water, 
and  the  water  then  mixed  with  the  gelatine,  as  many  of  the 
germs  are  taken  up  b}r  the  water. 

The  roll-cultures  of  Esmarch  give  the  best  results,  many  of 
the  varieties  usually  found  being  anaerobic. 

Animals  inoculated  with  the  soil  around  Berlin  die  almost 
always  of  malignant  oedema,  and  with  that  of  some  other  towns 
invariably  of  tetanus.  Many  of  the  germs  found  are  nitrogen 
formers  and  play  a  great  role  in  the  economy  of  the  soil. 

Nitrifying  organisms  are  found  in  the  superficial  layers  of  the 
earth.  Organic  matters  found  in  sewage  and  in  the  faecal  evac- 
uations of  animals  form  the  basis  for  their  activity,  whereby 
nitrates,  ammonias,  and  nitric  acid  result.  The  nitrogen  neces- 
sary for  the  growing  plant  is  thus  produced.  The  nitro-monas 
of  Winogradsky  belongs  to  this  group. 

The  Bacteria  of  Milk  and  Other  Foods.  Milk  as  secreted  is 
sterile,  but  at  every  step  in  its  passage  from  the  cow  to  the  con- 
sumer it  is  liable  to  contamination.  Even  the  lower  portion  of 
the  teat  is  a  source  of  infection,  owing  to  the  presence  of  stagnated 
milk  from  the  former  milking,  and,  as  consumed,  milk  usually 
contains  thousands  to  millions  of  bacteria  to  the  cubic  centimetre. 
Sterilization  or  Pasteurization  and  supervision  of  the  dairies 
should  always  be  carried  out  on  milk  used  for  infant  feeding. 

Foods  eaten  after  little  or  no  cooking,  such  as  fruits,  salads, 
and  the  like,  and  also  oysters,  are  possible  sources  of  bacterial 
diseases,  and  the  not  infrequent  so-called  ptomaine  poisoning 
observed  after  the  consumption  of  ice-cream,  sausage,  canned 
meats,  etc.,  is  the  result  of  the  action  of  bacteria  or  their 
products 


EXAMINATION     OF     THE     HUMAN     BODY.  ]  go, 


BACTERIOLOGIC   EXAMINATION   OF  THE   ORGANS   AND 
CAVITIES  OF  THE  HUMAN  BODY. 

The  body,  on  account  of  its  constant  contact  with  the  sur- 
rounding air,  is  necessarily  exposed  to  infection,  and  we  would 
be  likely  to  find  on  the  skin  and  in  the  oral,  anal,  and  nasal 
cavities  the  varieties  of  micro-organisms  commonly  around  us. 
Through  the  water  and  food  the  body  is  also  contaminated; 
but  some  organisms  by  predilection  inhabit  the  mouth,  intes- 
tine, and  other  cavities,  and  form  there  a  flora  distinctly  their 
own. 

The  Skin.  The  majority  of  micro-organisms  met  with  on  the 
skin  are  non-pathogenic,  although  underneath  the  nails  and  in 
the  hair,  pus-forming  micro-organisms  often  occur,  producing 
sometimes  serious  abscesses. 

In  the  sweat-glands  and  the  sebaceous  glands  various  organ- 
isms have  been  found.  The  Staphylococcus  epidermidis  albus 
of  Welch  is  present  normally. 

In  foul-smelling  perspiration  of  the  feet  Rosenbach  found 
Saprogenes  No.  II.,  which  is  pathogenic  for  rabbits. 

Micrococcus  cereus  albns  and  flavus,  Diplococcus  liquefa- 
ciens  albus  and  flavus,  Staphylococcus  pyogenes  aureus,  and 
Streptococcus  pyogenes  are  found  underneath  the  nails. 

In  eczema,  Diplococcus  albicans  tardus,  D.  citreus  liquefa- 
ciens,  D.  flavus  liquefaciens,  and  Ascobacillus  citreus. 

In  colored  sweat,  Micrococcus  hsematodes,  Bacillus  pyocya- 
neus. 

A  diplococcus  is  found  in  acute  pemphigus. 

The  lepra  bacillus,  the  tubercle  bacillus  in  lupus,  and  the 
typhoid  bacillus  in  the  eruption  of  typhoid  fever  are  a  few  of 
the  specific  germs  found  on  the  skin. 

The  Conjunctiva.  The  micrococcus  of  trachoma,  the  Koch- 
Weeks  bacillus,  considered  to  be  the  specific  cause  of  acute 
catarrhal  conjunctivitis,  or  "  pink  eye,"  and  the  Bacillus  xero- 
sis, are  special  germs  found  on  the  conjunctiva;  the  other  va- 
rieties of  air-  and  water-organisms,  and  those  usually  present 
on  the  skin,  are  also  found. 


190  APPENDIX. 

The  Mouth.  The  mouth  is  a  favorite  seat  for  the  development 
of  bacteria.  The  alkaline  saliva,  the  particles  of  food  left  in 
the  teeth,  the  decayed  teeth  themselves,  all  furnish  suitable 
soil  for  their  growth. 

Quite  a  number  of  germs  have  been  isolated  and  their  prop- 
erties partly  studied.  Many  have  some  connection  with  the 
production  of  caries  of  the  teeth,  as  Miller  has  well  shown  in 
his  careful  studies.  The  Leptothrix  buccalis,  found  in  nearly 
all  mouths,  is  a  long  chain  or  filamentous  bacillus  which  stains 
blue  with  iodin.  It  was  formerly  considered  the  cause  of  tartar 
on  the  teeth. 

The  Spirillum  sputigenum,  Spirochaeta  dentium,  Micrococcus 
gingivae  pyogenes,  Bacillus  dentalis  viridans,  B.  pulpae  pyogenes, 
Microccocus  of  sputum-septicaemia,  and  M.  salivarus  septicus 
are  a  few  of  the  germs  cultivated  by  Miller  and  Biondi  from 
the  mouth.  Besides  these,  the  pneumo-bacteria,  diphtheria 
bacillus,  and  tubercle  bacillus  are  often  met  with,  the  first  two 
in  the  mouths  of  healthy  persons.  The  expired  air  in  quiet 
respiration  is  free  from  bacteria,  but  in  coughing,  sneezing,  etc., 
large  numbers  of  organisms  are  violently  ejected  and  the 
atmosphere  about  tubercular  patients  is  always  saturated  with 
tubercle  bacilli. 

Ear.  In  the  middle  ear  of  new-born  infants  no  pathogenic 
organisms  were  found,  but  quite  a  number  of  non-pathogenic 
ones.  In  affections  of  the  ear  the  pneumo-bacillus  and  the 
Staphylococcus  pyogenes  are  most  frequent. 

Nasal  Cavity.  The  nasal  secretion,  containing  as  it  does 
dead  cells  and  being  alkaline  in  reaction,  forms  a  good  soil  for 
the  growth  of  germs. 

Diplococcus  coryzae,  Micrococcus  nasalis,  Bacillus  fcetidus 
ozaenae,  B.  striatus  albus  et  flavus,  B.  capsulatus  mucosus,  and 
Vibrio  nasalis  are  some  of  the  organisms  described  by  various 
observers. 

Stomach  and  Intestine.  The  secretion  of  the  stomach  is  in 
its  normal  state  not  a  favorable  soil  for  the  development  of 
bacteria,  yet  some  germs  resist  the  action  of  the  gastric  juice 
and  flourish  in  it.  When  the  acids  of  the  stomach  are  dimin- 
ished in  quantity  or  absent  altogether,  the  conditions  for  the 


EXAMINATION    OF    THE    HUMAN    BODY.  191 

growth  of  bacteria  are  more  favorable.  The  alimentary  canal 
of  the  newly  born  infant  is  sterile,  but  in  a  few  hours  micro- 
organisms begin  to  appear. 

Some  gastric  bacteria  normally  present  are  Sarcina  ventric- 
uli,  Bacterium  lactis  aerogenes,  Bacillus  subtilis,  B.  amylo- 
bacter,  B.  megaterium. 

The  intestinal  organisms  are  more  numerous,  and  the  mu- 
cous lining  of  the  intestines  and  the  secretions  there  present 
are  favorable  to  germ-growth. 

Bacillus  geniculates,  Boas  considers  a  sign  of  carcinoma  of 
the  stomach,  and  is  always  present,  he  claims,  when  the  con- 
tents contain  lactic  acid. 

Some  investigators  consider  digestion  dependent  on  microbic 
activity,  but  experiments  with  animals  have  recently  shown 
that  life  and  digestion  can  proceed  in  a  perfectly  sterile  condi- 
tion. Food  and  air  sterilized  will  not  develop  bacteria  in  the 
faeces. 

In  the  faeces  of  the  young  a  great  many  bacteria  have  been 
found  that  are  supposed  to  stand  in  close  relation  with  the 
intestinal  disorders  common  to  nurslings.  The  majority  of 
bacteria  usually  present  in  the  intestines  are  non-pathogenic. 
The  following  varieties  may  be  met  with  in  the  feces :  Micro- 
coccus aerogenes,  Bacillus  subtilis,  B.  butyricus,  B.  putrificus 
coli,  B.  lactis  aerogenes,  B.  coli  commune,  B.  subtiliformis,  and 
the  bacteria  of  cholera,  dysentery,  and  typhoid,  besides  many 
yeast-cells. 

Genito-urinary  Passages.  In  vaginal  secretion  Bumm  has 
been  able  to  find  a  number  of  organisms,  some  of  which 
closely  resemble  the  gonococcus;  thus,  there  is  the  Diplococ- 
cus  subflavus,  Micrococcus  lacteus  faviformis,  Diplococcus  albi- 
cans amplus,  and  the  vaginal  bacillus. 

In  the  urethra  of  healthy  persons  bacteria  are  sometimes 
found,  usually  having  entered  from  the  air. 

In  the  normal  secretions  around  the  prepuce  a  bacillus  called 
the  smegma  bacillus  has  been  discovered,  and  it  is  considered 
identical  with  the  so-called  syphilis  bacillus  of  Lustgarten. 

In  urethral  pus  a  number  of  diplococci  other  than  the  gono- 
cocci  have  been  isolated. 


192  APPENDIX. 

From  the  urine  itself  a  great  number  of  bacteria  have  been 
obtained,  but  mostly  derived  from  the  air,  finding  in  the  urine 
a  suitable  soil.  A  description  of  uro-bacteria  will  be  found  on 
page  91. 

Micro-organisms  of  the  Blood.  Many  of  the  bacteria  de- 
scribed in  the  body  of  this  book  are  found  in  the  blood  of  the 
animal  they  infect ;  thus,  anthrax  bacilli  are  always  found  in 
the  blood,  whereas  tubercle  bacilli  seldom,  if  ever,  enter  this 
secretion. 

When  animals  are  subcutaneously  injected  with  pneumo- 
cocci  they  are  found  in  large  quantities  in  the  blood.  The  dis- 
eases of  a  hemorrhagic  nature  affecting  fowls  and  swine  usually 
show  the  presence  of  bacteria  in  the  vascular  system. 

Bacteria  may  be  recovered  from  the  blood  in  all  forms  of 
septic  infection,  such  as  general  sepsis,  malignant  endocarditis, 
and  puerperal  sepsis. 

Method  of  Examination. — A  drop  of  blood  can  be  spread  on 
a  cover-glass  and  stained  with  the  ordinary  dyes,  as  sputum, 
pus,  or  serum ;  but  in  order  to  eliminate  the  coloring  matter 
of  the  red  corpuscles  and  bring  the  stained  bacteria  more 
prominently  into  view,  Gunther  recommends  that  the  blood, 
after  drying  and  fixing,  should  be  rinsed  in  a  dilute  solution 
of  acetic  acid  (1  to  5  per  cent.).  The  haemoglobin  is  thereby 
extracted,  and  the  corpuscles  appear  then  only  as  faint  out- 
lines. 

Instead  of  "  fixing  "  by  heat,  Canon  employs  alcohol  for  five 
minutes,  especially  in  staining  for  influenza  bacilli,  which  have 
been  detected  in  the  blood. 

This  method,  however,  requires  the  presence  of  enormous 
numbers  of  bacteria  in  order  to  succeed,  and  the  plan  com- 
monly employed  consists  in  making  "  blood  cultures."  As 
large  a  quantity  of  blood  as  possible — never  less  than  10  c.c. — 
is  taken  from  a  superficial  vein,  the  median  basilic,  for  ex- 
ample, by  means  of  a  sterile  antitoxin  syringe,  a  small  incision 
being  made  through  the  skin  over  the  vein  in  order  to  avoid 
skin  infection.  The  blood  so  obtained  is  immediately  trans- 
ferred to  culture  tubes,  which  are  then  studied  in  the  custom- 
arv  manner. 


PLATE  IV. 


-'  <  h  !  y 


i . 


'  \> 


H  I 


o 


BACILLI  OF  SYMPTOMATIC  ANTHRAX,   WITH   SPORES   iooo* 
(Frankel  and  Pfeiffer.) 


DIPHTHERIA  BACILLUS  PURE  CULTURE  ioooX- 
(Frankel  and  Pfeiffer.) 


PLATE  V. 


PFEIFFER'S  CAPSULE  BACILLUS  IN   BLOOD  ioooX- 
'Frankel  and  Pfeiffer.) 


YEAST-CELUS  500 X- 
(FrSnkel  and  Pfeiffer.) 


PLATE  VI. 


PENICILLIUM  GLAUCUM  500  X. 
(Frankel  and  Pfeitter.) 


ASPERGILLUS  FUMIGATUS  500  X- 
(Frankel  and  Pfeiffer.) 


INDEX 


Abbe's  condenser,  27 

Achorion  Schonleinii,  176 

Actinomyces,  177 

Actinomycosis,  177 

Aerobin,  24 

.Kstivo-autumnal  form  of  malarial 

parasite,  161 
Agar-agar,  54 

bouillon,  54 

glycerine,  55 
Agglutination  reaction  for  tubercle 

bacillus,  106 
Agglutinins,  7G 
Air,  examination  of,  180 
Alexin,  75 
Amboceptor,  75 
Amoeba  dysenteriae,  163 
Anaerobin,  24 
Anilin  dyes,  30 

oil,  31 

oil  water,  31 
Animals  for  experiment,  76 

tuberculosis  in,  105 
Anopheles,  159 
Anthrax,  94 
Anthraxin,  97 
Antitoxin  of  diphtheria,  113 

of  pneumonia,  134 

of  tetanus,  149 
Antituberculous  serum,  106 
Arnold's  sterilizer,  45 
Arthrospores,  22 
Asexual  cycle  in  man,  160 
Aspergillus  fumigatus,  177 

glaucus,  177 
Autoclave  of  Chamberland,  46 

Bacillus  acidilactici,  84 
aerogenes  capsulatus,  158 


Bacillus,  alvei,  169 
amylobacter,  85 
anthracis,  94 
avicidis,  165 
Boas-Oppler,  93 
butyricus,  85 
capsule,  136 
coeruleus,  88 
coli  communis,  122 
comma,  124 
erythrosporus,  88 
fluorescens,  142 
fluorescens  liquefaciens,  88 
geniculatus,  93 
icteroides,  154 
indicus,  81 
Klebs-Loffler,  110 
lactis  cyanogenus,  86 

erythrogenes,  86 
lepra,  107 
mallei,  108 
megaterium,  82 
melittophtharus,  169 
mesentericus  vulgatus,  81 
Milzbrand,  94 
murisepticus,  168 
mycoides,  82 
cedematis  maligni,  150 
of  American  swine  plague,  167 
of  anthrax,  94 
of  bluish-green  pus,  142 
of  bubonic  plague,  155 
of  cattle  plague,  167 
of  chicken  cholera,  165 
of  diphtheria,  110 

pseudo-,  114 
of  dysentery,  157 
of  French  swine  plague,  167 
of  glanders,  108 

237 


238 


INDEX. 


Bacillus  of  hog  cholera,  167 

of  influenza,  137 

of  malignant  oedema,  150 

of  mouse  septicaemia,  1 68 

of  ox  plague,  167 

of  rabbit  septicaemia,  167 

of  soft  chancre,  153 

of  steer  plague,  167 

of  Swedish  swine  plague,  167 

of  swine  erysipelas,  167 

of  symptomatic  anthrax,  164 

of  syphilis,  108 

of  tetanus,  147 

of  typhoid  fever,  115 

paracolon,  121 

paratyphoid,  121 

phosphorescens  gelidus,  89 
indicus,  89 
indigenus,  89 

pneumo-,  131 

potato,  81 

prodigiosus,  80 

pseudo-diphtheria,  114 

psittacosis,  121 

pyocyaneus,  142 
P-,  143 

ramosus,  82 

smegma,  108 

spinosus,  84 

subtilis,  83 

tuberculosis,  97 
products  of,  106 

violaceus,  87 
Bacteria,  17 

action  in  causing  disease,  70 

asporogenic,  22 

effect  on  body,  71 

fluorescent,  88 

higher,  17 

in  air,  183 

in  milk,  84 

in  urine,  91 

in  water,  87,  184 

infective,  71 

life  of,  23 

lower,  17 

non-pathogenic,  80,  183 

of  food,  188 

of  hemorrhagic  septicaemia,  166 


Bacteria  of  milk,  188 

of  pneumonia,  130 

origin  of,  23 

pathogenic,  25,  70,  94,  183 

persistence  in  water,  121 

phosphorescent,  89 

pyogenic,  71 

similar  to  cholera,  127 

staining  of,  30 

structure  of,  18 

suppurative,  71 

tables  of,  194-235 

toxic,  70 

unstained,  27 

vital  actions  of,  24 
Bactericie  du  charbon,  94 
Bacteriolysis,  75 
Bacterium  acidi  lactici,  84 

aeruginosum,  142 

Balticum,  90 

Fischeri,  90 

Pflugeri,  90 

syncyanum,  86 

termo-,  170 

ureae,  91 

zopfi,  83 
Beef  extract,  48 
Beggiatoa  alba,  90 
Behavior   of    bacteria   to    Gram' 

stain,  41 
Benches  for  glass  plates,  63 
Biedert's  method.  103 
Black-leg,  164 
Blight,  170 
Blood  cultures,  192 
Blood-coagulum,  58 
Blood-serum  as  media,  55 
Boas-Oppler  bacillus,  93 
Botkin's  method,  69 
Bouillon,  48 

gelatine,  52 

guinea-pig,  59 

preparation  of,  48 
Bovine  tuberculosis,  105 
Bowhill's  orcein  stain,  40 
Bread  mash,  51 
Brood-oven,  56 
Brownian  movements,  19 
Buchner's  method,  69 


INDEX. 


239 


CArsuLE  stain  of  Hiss,  35 

of  Welch,  35 
Carbol-thionin  solution,  34 
Cattle  plague,  167 
Cell  contents,  18 

wall,  18 
Celloidin  sacs,  79 
Cellular  theory,  74 
Charbon  symptomatique,  164 
Charcoal  niter,  185 
Chemotaxis,  74 
Cholera,  124 

Pfeitfer's  immunity,  127 
Cladothrices,  177 
Cladothrix  actinomyces,  177 

dichotoma,  90 
Clostridium,  21 
-  butyricum,  85 
Complement,  75 
Cotton  plugs,  47 
Cover-glass  preparations,  35 
Crenothrix,  90 

Kulmiana,  90 
Cultivation,  42 

artificial,  42 

methods  of,  42 

of  anaerobins,  67 
Cultures,  appearances  of,  64 

egg,  59 

glass-plate,  62 

glass-slide,  60 

potato,  49 

rolled,  64 

test-tube,  60 
Cytase,  75 

Dkcolorants,  32 
Diphtheria,  110 

Diplococcus  albicans  ampins,  146 
tardissimus,  146 

lanceolatus,  132 

of  meningitis,  135 

of  pneumonia,  130 
Disinfectants,  42 
Drying  specimens,  35 
Dunham's    rosalic    acid     solution, 
59 

peptone  solution,  125 
Dysentery,  157 


Effect  of  age,  26 
Ehrlich's  side-chain  theory,  75 
Eisner's  medium,  59 
Endospores,  21 
Enteric  fever,  115 
Esmarch's  method,  67  • 

tubes,  64 
Examination      of     human     body, 

189 
Experiments  on  animals,  76 

Favus,  176 

Ferments,  25,  71 
Filters,  185 

hot-water,  53 

Pasteur-Chamberland,  185 

sand,  182 
Fishing,  66 
Fission,  20 
Fission-fungi,  17 
Flagella,  19 

staining  of,  41 
Fluorescence,  26 
P'ood,  bacteria  of,  188 
Foul-brood,  169 
Franker s  borer,  187 

method  for  anaerobins,  68 

stain  for  tubercle  bacillus,  101 
Frog  plague,  167 
Fuchsin,  carbol-,  31,  33 
Fungi,  173 
Fungus,  rav,  177 

thrush,  174 

Gabbett's  stain,  34,  101 
Gametes,  160 
Gas  formation,  26 
Gelatine,  52 

bouillon,  52 

carbolized,  117 

paste,  37 

plates,  170 
Gelatinous  membrane,  18 
Germination,  22 
Glucose  broth,  49 
Glycerin  broth,  49 
Gonococcus,  143 
Gonorrhoea,  143 
Gonotoxin,  145 


240 


INDEX 


Gram's  stain,  33 

Gruber-Widal  blood-serum  test,  117 

HjEMAMCEBA  malarise,  159 

vivax,  159 
Hsemomenas  prcecox,  160  , 
Hanging-drop,  29 
Haptins,  75 
Haptophores,  75 
Heat  as  disinfectant,  43 

dry,  43 

moist,  44 
Hemorrhagic  septicaemia,  166 
Herpes  tonsurans,  176 
Hesse's  method  for  air,  180 

for  anaerobins,  67 
Hiss'  capsule  stain,  35 

typhoid  medium,  59 
Hog  cholera,  167 
Homogeneous  lens,  26 
Hot-air  oven,  43 
Hot- water  filter,  53 
Hiippe's  method,  68 

Immersion  lens,  26 
Immune  body,  75 
Immunity,  72 

theories  of,  74 
Incubators,  56 
Infection,  70 

conditions  necessary  to  produce, 
70   > 
Inoculation  of  animals,  76 
Iodin,  32 
Iris  blender,  27 
Iron  box  for  plates,  62 

Japanese  method,  55 
Jenner's  stain,  162 

Klatsch  preparations,  66 
Koch's  rules,  79 

stain,  33 

steam-chest,  44 
Kiihne's  method,  41 

stain,  33 

Leprosy,  107 
Leptothrix  buccalis,  90 


Liborius's   method  for  anaerobins, 

67 
Locomotion,  19 
Loftier' s  alkaline  stain,  33 

blood  serum,  111 

mordant,  33 
Lysins,  75 

Macrocytases,  75 
Macrogamete,  161 
Macrophages,  74 
Malarial  parasite,  159 
Malignant  oedema,  150 
Mallein,  110 

Marchoux's  thionin  stain,  162 
Marmorek's  serum,  106 
Material  from  animals,  79 
Media,  nutrient,  48 

solid,  49 

transparent,  52 
Merozoites,  160 
Metchnikoff ' s  theory,  74 
Microbe  en  huit,  165 
Micrococci  similar  to  gonococcus, 

145 
Micrococcus  amylovorus,  170 

cereus  albus,  141 
flavus,  141 

cholera  gallinarum,  165 

citreus  conglomeratus,  145 

indicus,  81 

melitensis,  159 

of  gonorrhoea,  143 

of  mal  de  pis,  169 

of  sputum  septicaemia,  132 

Pasteuri,  132 

pyogenes  aureus,  141 
citreus,  141 
tenuis,  142 

subflavus,  146 

tetragenus,  135 

ureae,  91 
Microcytases,  75 
Microgametes,  160 
Microgametocytes,  160 
Micro-organisms    of    suppuration, 

137 
Microphages,  74 
Microscope,  26 


NDEX. 


241 


Microsporon  furfur,  176 

Milzbrand,  94 

Moist  chamber,  50 

Mordants,  31 

Mosquito,  sexual  cycle  in,  160 

Moulds,  175 

examination  of,  177 
Mouse  septicaemia,  168 
Movements,  vibratory,  19 
Mucor  mucedo,  175 
Mycoprotein,  18 

Nail  culture,  132 
Neisser's  stain,  34 
Nicolle's  solution,  34 
Nitrification,  25 
Nitromonas,  188 
Nivellier  apparatus,  62 
Nutrient  media,  48 

Odors  in  cultures,  25 
Oi'dium,  174 

albicans,  174 

lactis,  174 
Oil  immersion,  26 
Orcein  stain,  40 
Oxidation,  25 

Parasites,  23 

malarial,  159 
Park's  method,  69 
Pasteur  filter,  185 
Pathogenic  yeasts,  174 
Penicillium  glaucum,  175 
Peptone  solution,  58 
Dunham's,  125 
Petri's  sand  Altera,  182 

saucer,  63,  186 
Pfeiffer's  immunity  from  cholera, 

127 
Phagocytic  theory,  74 
Phosphorescence,  25 
Pigmentation,  25 
Platinum  needles,  28 
Pneumo-bacillus,  131,  132 
Potato  cubes,  51 

cultures,  49 

inoculation  of,  51 

in  test-tubes,  51 
16 


Potato  mash,  51 

Precipitins,  76 

Products  of  tubercle  bacilli,  106 

Proteins,  25 

Proteus,  170 

mirabilis,  171 

vulgaris,  170 

Zenkeri,  171 
Protozoa,  pathogenic,  159 
Pseudo-diphtheria  bacilli,  114 
Ptomaines,  25,  71 
Putrefaction,  25 
Pyocyanin,  143 
Pyrosoma  bigeminum,  163 

Quartan  forms  of  malarial  para- 
site, 161 

Rabbit  septicaemia,  167 
Rauschbrand,  165 
Ray  fungus,  177 
Receptors,  75 
Reduction,  25 
Relapsing  fever,  152 
Removing  excess  of  stain,  36 
Reproduction,  20 
Rosalie  acid  solution,  59 
Rotz,  108 

Rouget  du  pore,  167 
Roux's  stain,  34 
test-tube,  51 

Saccharomyces  albicans,  173 

cerevisiae,  173 

mycoderma,  174 

niger,  173 

rosaceus,  173 
Sapremia,  71 
Saprophytes,  23 
Sarcina,  92 

alba,  93 

aurantica,  93 

flava,  93 

lutea,  92 

rosea,  93 

ventriculi,  93 
Schizomycetes,  17 
Sehizophyceae,  17 
Schizophyta,  17 


242 


INDEX. 


Schultz's  method,  49 

Schweinerotlauf,  167 

Sedge  wick-Tucker  method,  183 

Septicemia,  71 

Serum,  antituberculous,  106 

test,  125 
Serum-agar,  111 
Sexual  cycle  in  mosquito,  160 
Slides,  concave,  29 
Small-pox,  163 
Soil,  examination  of,  187 
Solutions,  composite,  31 

formulae  of,  32 

saturated,  32 

stock,  31,  32 

weak,  31,  32 
Soor,  174 
Spasmotoxin,  149 
Specimens,  cover-glass,  35 

cutting  of,  37 

drying  of,  35 

Klatsch,  66 
Spirillum,  18 

cholera?,  124 

concentricum,  92 

Finkleri,  127 

of  relapsing  fever,  152 

rubrum,  92 

tyrogenum,  128 
Spirochete  Obermeieri,  152 
Spores,  arthro-,  21,  22 

contents  of,  21 

endo-,  21,  22 

formation  of,  21,  22 

requisites  for,  22 

resistance  of,  22 

staining  of,  40 
Sporidium  vaccinale,  163 
Sputum,  hardened,  104 
Stain,  alkaline,  31,  33 

aniline-water,  32 

chloroform  methyl-blue,  87 

Gabbett's,  34 

Gram's,  33 

Hiss',  35 

Koch's,  33 

Kiihne's,  33 

Loffler's,  33 

Neisser's,  34 


Stain,  Roux's,  34 

Unna's,  33 

Welch's,  35 

Ziehl-Neelsen,  33 
Staining,  Ernst's  method  of,  41 

general  method  of,  35 

Gram's  method  of,  39 

Jenner's  method  of,  162 

Kiihne's  method  of,  41,  109 

Loffler's  method  of,  109 

Marchoux's  method  of,  162 

of  capsule  of  bacillus  of  pneu- 
monia, 132 

of  flagella,  41 

of  malarial  parasite,  162 

of  milk,  87 

of  spores,  40 

of  sporogenic  bodies,  41 

of  tissue  sections,  37,  39 

rapid  method  of,  for  bacillis  tu- 
berculosis, 101 

slow  method  of,  for  bacillus  tu- 
berculosis, 102 

solutions,  31 

special  methods  of,  39 

Weigert's  method  of,  41 

Wright's  method  of,  162 
Staphylococcus,  18 

pyogenes  albus,  141 
aureus,  140 

epidermidis  albus,  141 
Sterilization,  42 

fractional,  47 
Streptococcus,  18 

erysipelatis,  138 

puerperalis,  139 

pyogenes,  138 
Streptothrix,  177 

farcinica,  179 

Madura,  178 
Suppuration,  137 
Swine  erysipelas,  167 

plague,  167 

Tertian   form  of  malarial   para- 
site, 161 
Test-tubes,  47 
Tetanin,  119 
Tetanotoxin,  149 


INDEX 


243 


Tetanus,  147 
Thermo-regulator,  58 
Thread  reaction,  76 
Thrush,  174 
Tinea,  176 

Tissue  preparations,  37 
Toxalbumins,  25,  71 
Toxins,  25,  71 

nature  of,  71 
Toxophores,  75 
Trichophyton  tonsurans,  176 
Trypanosomes,  163 
Tuberculin,  106 
Tuberculin  E,  106 
Tuberculocidin,  106 
Tuberculosis,  97 

in  animals,  105 
Typhoid  fever,  115 

medium  of  Hiss,  59 
Typhotoxin,  121 

Urine  media,  59 

Vaccinia,  163 


Vibrio,  Finkler-Prior,  127 

Metschnikovi,  129 
Vibrion  septique,  150 

Water,  bacteria  in,  87,  127,  180 
examination  of,  184 
persistence  of  bacilli  in,  121 

Weigert's  method  of  staining,  41 

Welch's  capsule  stain,  35 

Widal's  serum  test,  125 

Wild  plague,  167 

Wire  cages,  47 

Wolf hiigel's  apparatus,  186 

Wright's  chromatin  stain,  162 
method,  69 

Yeasts,  173 

examination  of,  177 

pathogenic,  174 
Yellow  fever,  etiology,  155 

Ziehl's  solution,  33 
Zoogloea,  18 


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and  complete  dictionary  of  the  terms  used  in  Medicine,  Surgery, 
Dentistry,  Pharmacy,  Chemistry,  and  kindred  branches;  with 
over  ioo  new  and  elaborate  tables  and  many  handsome  illustra- 
tions. By  W.  A.  Newman  Dorland,  M.  D.,  Editor  of  "The 
American  Pocket  Medical  Dictionary. "  Large  octavo,  850  pages, 
bound  in  full  flexible  leather.  Price,  $4.50  net;  with  thumb 
index,  $5.  00  net. 

Gives  a  Maximum  Amount  of  Matter  in  a  Minimum  Space,  and  at  the 
Lowest  Possible  Cost 

WITH   2000  NEW  TERMS 

The  immediate  success  of  this  work  is  due  to  the  special  features  that 
distinguish  it  from  other  books  of  its  kind.  It  gives  a  maximum  of  matter 
in  a  minimum  space  and  at  the  lowest  possible  cost.  Though  it  is  practi- 
cally unabridged,  yet  by  the  use  of  thin  bible  paper  and  flexible  morocco 
binding  it  is  only  i%  inches  thick.  In  this  new  edition  the  book  has 
been  thoroughly  revised,  and  upward  of  two  thousand  new  terms  have 
been  added,  thus  bringing  the  book  absolutely  up  to  date.  The  book  con- 
tains hundreds  of  terms  not  to  be  found  in  any  other  dictionary,  over  ioo 
original  tables,  and  many  handsome  illustrations. 


PERSONAL  OPINIONS 


Howard  A.  Kelly,  M.  D., 

Professor  of  Gynecology,  Johns  Hopkins  University,  Baltimore. 

"  Dr.  Dorland's  dictionary  is  admirable.  It  is  so  well  gotten  up  and  of  such  conve- 
nient size.     No  errors  have  been  found  in  my  use  of  it." 

Roswell  Park,  M.  D., 

Professor  of  Principles  and  Practice  of  Surgery  and  of  Clinical  Surgery,  University 
of  Buffalo. 

"  I  must  acknowledge  my  astonishment  at  seeing  how  much  he  has  condensed  within 
relatively  small  space.  I  find  nothing  to  criticize,  very  much  to  commend,  and  was  inter- 
ested in  finding  some  of  the  new  words  which  are  not  in  other  recent  dictionaries." 


PERSONAL  HYGIENE. 


Galbraith's 
Four  Epochs  qf  Woman's  Life 

Second  Revised  Edition — Recently  Issued 


The  Four  Epochs  of  Woman's  Life :  A  Study  in  Hygiene. 
By  Anna  M.  Galbraith,  M.  D.,  Fellow  of  the  New  York  Acad- 
emy of  Medicine,  etc.  With  an  Introductory  Note  by  John  H. 
Musser,  M.  D.,  Professor  of  Clinical  Medicine,  University  of 
Pennsylvania.      121110  volume  of  247  pages.     Cloth,  $1.50  net. 

In  this  instructive  work  are  stated,  in  a  modest,  pleasing,  and  conclusive 
manner,  those  truths  of  which  every  woman  should  have  a  thorough  knowl- 
edge. Written,  as  it  is,  for  the  laity,  the  subject  is  discussed  in  language 
readily  grasped  even  by  those  most  unfamiliar  with  medical  subjects. 

Birmingham  Medical  Review,  England 

"  We  do  not  as  a  rule  care  for  medical  books  written  for  the  instruction  of  the  public. 
But  we  must  admit  that  the  advice  in  Dr.  Galbraith's  work  is  in  the  main  wise  and  whole- 


Pyle's  Personal  Hygiene 


A  Manual  of  Personal  Hygiene :  Proper  Living  upon  a 
Physiologic  Basis.  By  Eminent  Specialists.  Edited  by  Walter 
L.  Pyle,  A.  M.,  M.  D.,  Assistant  Surgeon  to  Wills  Eye  Hospital, 
Philadelphia.  Octavo  volume  of  441  pages,  fully  illustrated. 
Cloth,  $1.50  net. 

NEW  (2d)  EDITION— RECENTLY  ISSUED 

In  this  new  second  edition  there  have  been  added  new  chapters  on  Home 
Gymnastics  and  Domestic  Hygiene,  besides  an  Appendix  of  Emergency  Pro- 
cedures. 

Boston  Medical  and  Surgical  Journal 

"The  work  has  been  excellently  done,  there  is  no  undue  repetition,  and  the  writers  have 
succeeded  unusually  well  in  presenting  facts  of  practical  significance  based  on  sound 
knowledge." 


SAUNDERS'    BOOKS   ON 


Draper's  Legal  Medicine 

A  Text-Book  of  Legal  Medicine.  By  Frank  Winthrop 
Draper,  A.  M.,  M.  I).,  Professor  of  Legal  Medicine  in  Harvard 
University,  Boston.  Handsome  octavo  of  573  pages,  illustrated. 
Cloth,  $4-oo  net. 

RECENTLY  ISSUED 

The  author  of  this  work  has  had  twenty -six  years'  experience  as  Medical 
Examiner  for  the  city  of  Boston,  his  investigations  comprising  nearly  eight 
thousand  deaths  under  a  suspicion  of  violence. 

Hon.  Olin  Bryan,  LL.  B. 

Professor  of  Medical  Jurisprudence,  Baltimore  Medical  College 
"  It  is  comprehensive,  thorough,  and  must,  of  a  necessity,  prove  a  splendid  acquisition 
to  the  libraries  of  those  who  are  interested  in  medical  jurisprudence." 

Jakob  arid  FisherV 
Nervous  System  and  its  Diseases 

Atlas  and  Epitome  of  the  Nervous  System  and  its 
Diseases.  By  Professor  Dr.  Chr.  Jakob,  of  Erlangen.  From 
the  Second  Revised  German  Edition.  Edited,  with  additions,  by 
Edward  D.  Fisher,  M.  D.,  Professor  of  Diseases  of  the  Nervous 
System,  University  and  Bellevue  Hospital  Medical  College,  New 
York.  With  83  plates  and  copious  text.  Cloth,  $3.50  net.  In 
Saunders'  Hand- Atlas  Series. 

The  matter  is  divided  into  Anatomy,  Pathology,  and  Description  of  Dis- 
eases of  the  Nervous  System.  The  plates  illustrate  these  divisions  most 
completely  ;  especially  is  this  so  in  regard  to  pathology.  The  exact  site  and 
character  of  the  lesion  are  portrayed  in  such  a  way  that  they  cannot  fail  to 
impress  themselves  on  the  memory  of  the  reader. 

Philadelphia  Medical  Journal 

**  We  know  of  no  one  work  of  anything  like  equal  size  which  covers  this  important  and 
complicated  field  with  the  clearness  and  scientific  fidelity  of  this  hand-atlas." 


DISEASES    OF  CHILDREN.  g 

American  Text-Book  of 
Diseases  of  Children 

American  Text-Book  of  Diseases  of  Children.  Edited 
by  Louis  Starr,  M.  I).,  Consulting  Pediatrist  to  the  Maternity 
Hospital,  etc.  ;  assisted  by  Thompson  S.  Westcott,  M.  D., 
Attending  Physician  to  the  Dispensary  for  Diseases  of  Children, 
Hospital  of  trie  University  of  Pennsylvania.  Handsome  octavo, 
1244  pages,  profusely  illustrated.  Cloth,  $7.00  net;  Sheep  or 
Half  Morocco,  $8.00  net. 

SECOND    REVISED    EDITION 

To  keep  up  witli  the  rapid  advances  in  the  field  of  pediatrics,  the  whole 
subject-matter  embraced  in  the  first  edition  has  been  carefully  revised,  new 
articles  added,  some  original  papers  amended,  and  a  number  entirely  rewrit- 
ten and  brought  up  to  date. 

British  Medical  Journal 

"  May  be  recommended  as  a  thoroughly  trustworthy  and  satisfactory  guide  to  the  subject 
of  the  diseases  of  children." 

Paul's  Fever  Nursing' 


Nursing  in  the  Acute  Infectious  Fevers.  By  George  P. 
Paul,  M.  D. ,  Assistant  Visiting  Physician  to  the  Samaritan  Hos- 
pital, Troy,  N.  Y.      121110  of  200  pages.     Cloth,  $1.00  net. 

JUST   ISSUED 

Dr.  Paul  has  written  his  book  especially  for  the  trained  nurse,  so  that  all 
extraneous  matter  has  been  studiously  avoided.  Great  stress  has  been  laid 
upon  care  and  management  in  each  disease,  as  this  relates  directly  to  the 
duties  of  the  nurse.  The  work  discusses  fever  in  general,  then  each  acute 
infectious  fever  separately,  and  finally  those  practical  procedures  necessary  to 
the  proper  management  of  the  fevers  described. 


io  SAUNDERS'  BOOKS  ON 

Friedenwald  &  Ruhrah's 
Dietetics  for  Nurses 


Dietetics  for  Nurses.  By  Julius  Friedenwald,  M.  D., 
Clinical  Professor  of  Diseases  of  the  Stomach,  College  of  Physi- 
cians and  Surgeons,  Baltimore;  and  John  Ruhrah,  M.  D., 
Clinical  Professor  of  Diseases  of  Children,  College  of  Physicians 
and  Surgeons,  Baltimore.     i2moof  363  pages.     Cloth,  $1.50  net. 

JUST  ISSUED 

This  work  has  been  prepared  to  meet  the  needs  of  the  nurse,  both  in  the 
training  school  and  after  graduation.  Rectal  alimentation  and  the  feeding 
of  operative  cases  are  fully  described. 

Edinburg  Medical  Journal. 

"  It  appears  to  us  to  contain  all  the  practical  side  of  dietetics,  of  handy  size  and  devoid 
of  padding." 


Lewis'  Anatomy  and 
Physiology  for  Nurses 


Anatomy  and  Physiology  for  Nurses.  By  LERoy  Lewi^ 
M.  D.,  Surgeon  to  and  Lecturer  on  Anatomy  and  Physiology  for 
Nurses  at  the  Lewis  Hospital,  Bay  City,  Michigan.  i2mo  of  317 
pages,  with  146  illustrations.     Cloth,  $1.75  net. 

JUST  ISSUED 

The  author  has  based  the  plan  and  scope  of  the  work  on  the  methods  he 
has  employed  in  teaching  the  subjects,  and  has  made  the  text  unusually 
simple  and  clear.  The  object  was  so  to  deal  with  anatomy  and  physiology  that 
the  student  might  easily  grasp  the  primary  principles,  at  the  same  time  laying 
a  broad  foundation  for  a  wider  study. 


NURSING.  1 1 


De  Lee's  Obstetrics  for  Nurses 

Obstetrics  for  Nurses.  By  Joseph  B.  De  Lee,  M.  D.,  Pro- 
fessor of  Obstetrics  in  the  Northwestern  University  Medical  School, 
Chicago  ;  Lecturer  in  the  Nurses'  Training  Schools  of  Mercy, 
Wesley,  Provident,  Cook  County,  and  Chicago  Lying-in  Hos- 
pitals.    121110  of  460  pages,  fully  illustrated.      Cloth,  $2.50  net. 

JUST  ISSUED— NEW  2nd  (EDITION 

The  illustrations  in  Dr.  De  Lee's  work  are  nearly  all  original,  and  repre- 
sent photographs  taken  from  actual  scenes.  The  text  is  the  result  of  the 
author's  eight  years'  experience  in  lecturing  to  nurses. 

J.  Clifton  Edgar,  M.  D., 

Professor  of  Obstetrics  and  Clinical  Midwifery,  Cornell  University,  Neiv  York. 
"  It  is  far  and  away  the  best  that  has  come  to  my  notice,  and  I  shall  take  great  pleasure 
in  recommending  it  to  my  nurses,  and  students  as  well." 


Davis'  Nursing 

Obstetric  and  Gynecologic  Nursing.  By  Edward  P. 
Davis,  A.  M.,  M.  D.,  Professor  of  Obstetrics,  Jefferson  Medical 
College,  Phila.    i2mo,  400  pages,  illustrated.   Buckram,  $1.75  net. 

RECENTLY  ISSUED— SECOND  REVISED  EDITION 
The  Lancet,  London 

"  Not  only  nurses,  but  even  newly  qualified  medical  men,  would  learn  a  great  deal  by  a 
perusal  of  this  book.  It  is  written  in  a  clear  and  pleasant  style,  and  is  a  work  we  can 
recommend." 

Beck's  Reference  Handbook 
for  Nurses 

A  Reference  Handbook  for  Nurses.  By  Amanda  K.  Beck, 
Chicago.     121110  of  150  pages.     Flexible  morocco,  $1.25  net. 

RECENTLY  ISSUED 

This  little  book  contains  information  upon  every  question  that  comes  to  a 
nurse  in  her  daily  work,  and  embraces  all  the  information  that  she  requires  to 
carry  out  any  directions  given  by  the  physician. 

Boston  Medical  and  Surgical  Journal 

"  Must   be   regarded  as  extremely  useful,  not  only  for  nurses,  but  for  physicians." 


SAUNDERS'    BOOKS   ON 


Hof matin  and  Peterson's 
Legal  Medicine 


Atlas  of  Legal  Medicine.  By  Dr.  E.  von  Hofmann,  of 
Vienna.  Edited  by  Frederick  Peterson,  M.  1).,  Clinical  Profes- 
sor of  Psychiatry  in  the  College  of  Physicians  and  Surgeons,  New 
York.  With  120  colored  figures  on  56  plates,  and  193  half-tone 
illustrations.    Cloth  $3.50  net.     In  Saunders''  Hand-Atlas  Series. 

By  reason  of  the  wealth  of  illustrations  and  the  fidelity  of  the  colored 
plates,  the  book  supplements  all  the  text-books  on  the  subject.  Moreover, 
it  furnishes  to  every  physician,  student,  and  lawyer  a  veritable  treasure-house 
of  information. 

The  Practitioner,  London 

"  The  illustrations  appear  to  be  the  best  that  have  ever  been  published  in  connection 
with  this  department  of  medicine,  and  they  cannot  fail  to  be  useful  alike  to  the  medical  jurist 
and  to  the  student  of  forensic  medicine." 

Chapman's 
Medical  Jurisprudence 


Medical  Jurisprudence,  Insanity,  and  Toxicology.     By 

Henry  C.  Chapman,  M.  D.,  Professor  of  Institutes  of  Medicine 
and  Medical  Jurisprudence  in  Jefferson  Medical  College,  Phila- 
delphia. Handsome  1 21110  of  329  pages,  fully  illustrated.  Cloth, 
$1.75  net. 

RECENTLY  ISSUED— THIRD  REVISED  EDITION,  ENLARGED 

This  third  edition  has  been  thoroughly  revised  and  greatly  enlarged,  so  as 
tc  bring  it  absolutely  in  accord  with  the  very  latest  advances  in  this  important 
branch  of  medical  science. 

Medical  Record,  New  York 

"The  manual  is  essentially  practical,  and  is  a  useful  guide  for  the  general  practitioner, 
besides  possessing  literary  merit." 


IfUSSING.  13 


Golebiewski  and  Bailey V 
Accident  Diseases 


Atlas  and  Epitome  of  Diseases  Caused  by  Accidents. 

By  Dr.  Ed.  Golebiewski,  of  Berlin.     Edited,  with  additions,  by 

Pearce    Bailey,   M.  D.,   Consulting  Neurologist  to   St.  Luke's 

Hospital,  New  York.     With  71  colored  illustrations  on  40  plates, 

143  text-illustrations,  and  549  pages  of  text.      Cloth,  $4.00  net. 

In  Saunders'  Hand- Atlas  Series. 

This  work  contains  a  full  and  scientific  treatment  of  the  subject  of  accident 
injury  ;  the  functional  disability  caused  thereby  ;  the  medicolegal  questions 
involved,  and  the  amount  of  indemnity  justified  in  given  cases. 

The  Medical  Record,  New  York 

"  This  volume  is  upon  an  important  and  only  recently  systematized  subject,  which  is 
growing  in  extent  all  the  time.     The  pictorial  part  of  the  book  is  very  satisfactory." 

StoneyV 
Materia  Medica  for  Nurses 


Practical  Materia  Medica  for  Nurses,  with  an  Appendix 
containing  Poisons  and  their  Antidotes,  with  Poison-Emergencies ; 
Mineral  Waters  ;  Weights  and  Measures,  etc.  By  Emily  M.  A. 
STONEY,  Superintendent  of  the  Training  School  for  Nurses  at  the 
Carney  Hospital,  South  Boston,  Mass.    1 2mo,  300  pages.   31.50  net. 

JUST    ISSUED— NEW  (3rd)  EDITION 

In  this  work  the  consideration  of  the  drugs  includes  their  names,  their 
sources  and  composition,  their  various  preparations,  physiologic  actions, 
directions  for  handling  and  administering,  and  the  symptoms  and  treatment 
of  poisoning. 

Journal  of  the  American  Medical  Association 

"  So  far  as  we  can  see.  it  contains  everything  that  a  nurse  ought  to  know  in  regard  to 
drugs.     As  a  reference-book  for  nurses  it  will  without  question  be  very  useful." 


14  SAUNDERS'  BOOKS   ON 

StoneyV  Nursing 

Practical  Points  in  Nursing :  for  Nurses  in  Private  Practice. 
By  Emily  M.  A.  Stoney,  Superintendent  of  the  Training 
School  for  Nurses  at  the  Carney  Hospital,  South  Boston,  Mass. 
466  pages,  fully  illustrated.     Cloth,  $1.75  net. 

THIRD   REVISED    EDITION— RECENTLY   ISSUED 


In  this  volume  the  author  explains  the  entire  range  of  private  nursing  as 
distinguished  from  hospital  nursing,  and  the  nurse  is  instructed  how  best  to 
meet  the  various  emergencies  of  medical  and  surgical  cases  when  distant 
from  medical  or  surgical  aid  or  when  thrown  on  her  own  resources.  An 
especially  valuable  feature  will  be  found  in  the  direction  how  to  improvise 
everything  ordinarily  needed  in  the  sick-room. 

The  Lancet,  London 

'*  A  very  complete  exposition  of  practical  nursing  in  its  various  branches,  including 
obstetric  and  gynecologic  nursing.      The  instructions  given  are  full  of  useful  detail." 


Stoney's  Technic  for  Nurses 

Bacteriology  and  Surgical   Technic  for   Nurses.      By 

Emily  M.  A.  Stoney,  Superintendent  of  the  Training  School> 
Carney  Hospital,  South  Boston.  Revised  by  Frederic  R.  Grif- 
fith, M.  D.,  Surgeon,  N.  Y.    i2mo,  278  pages,  illus.    #1.50  net. 

RECENTLY  ISSUED— NEW  (2d)  EDITION 

Spratling  on  Epilepsy 

Epilepsy  and  Its  Treatment.  By  William  P.  Spratling, 
M.  D. ,  Medical  Superintendent  of  the  Craig  Colony  for  Epilep- 
tics, Sonyea,  New  York.  Octavo  of  522  pages,  fully  illustrated. 
Cloth,  $4.00  net. 


CHILDREN  AND  HYGIENE.  15 

Griffiths 
Care  of  the  Baby 

The  Care  of  the  Baby.  By  J.  P.  Crozer  Griffith,  M.  D., 
Clinical  Professor  of  Diseases  of  Children,  University  of  Penn- 
sylvania.     i2mo,  436  pages.     Illustrated.     Cloth,  $1.50  net. 

RECENTLY   ISSUED— THIRD   EDITION,  REVISED 

New  York  Medical  Journal 

"  We  are  confident  if  this  little  work  could  find  its  way  into  the  hands  of  every  trained 
nurse  and  of  every  mother,  infant  mortality  would  be  lessened  by  at  least  fifty  per  cent." 

Crothers'  Morphinism 

Morphinism  and  Narcomania  from  Opium,  Cocain,  Ether, 
Chloral,  Chloroform,  and  other  Narcotic  Drugs ;  also  the  Etiol- 
ogy, Treatment,  and  Medicolegal  Relations.  By  T.  D.  Croth- 
ers, M.  D.,  Superintendent  of  Walnut  Lodge  Hospital,  Hartford, 
Conn.  Handsome  i2mo  of  351  pages.  Cloth,  $2.00  net. 
The  Lancet,  London 

"  An  excellent  account  of  the  various  causes,  symptoms,  and  stages  of  morphinism,  the 
discussion  being  throughout  illuminated  by  an  abundance  of  facts  of  clinical,  psychological, 
and  social  interest." 

Abbott's 
Transmissible  Diseases 

The  Hygiene  of  Transmissible  Diseases:  Their  Causa- 
tion, Modes  of  Dissemination,  and  Methods  of  Prevention.  By 
A.  C.  Abbott,  M.  D.,  Professor  of  Hygiene  and  Bacteriology, 
University  of  Pennsylvania.  Octavo,  35 1  pages,  with  numerous 
illustrations.     Cloth,  $2.50  net. 

SECOND  REVISED  EDITION 
The  Lancet,  London 

"  We  heartily  commend  the  book  as  a  concise  and  trustworthy  guide  in  the  subject  with 
which  it  deals,  and  we  sincerely  congratulate  Professor  Abbott." 


1 6  SAUNDERS*    BOOKS   ON  NUI&ING. 

American  Pocket  Dictionary  4th  Ed.— Recently  issued 

American  Pocket  Medical  Dictionary.  Edited  by  W.  A. 
Newman  Dorland,  M.  D.,  Assistant  Obstetrician  to  the  Hospital  of 
the  University  of  Pennsylvania.  Containing  the  pronunciation  and  defi- 
nition of  the  principal  words  used  in  medicine  and  kindred  sciences, 
with  64  extensive  tables.  Handsomely  bound  in  flexible  leather,  with 
gold  edges,  $  I. 00  net;  with  patent  thumb  index,  #1.25  net. 

Morrow's  Immediate  Care  of  Injured      Just  Ready 

Immediate  Care  of  the  Injured.  By  Albert  S.  Morrow,  M.  D., 
Attending  Surgeon  to  the  New  York  City  Hospital  for  the  Aged  and  In- 
firm.    Octavo  of  350  pages,  with  250  illustrations. 

Dr.  Morrow's  book  on  emergency  procedures  is  written  in  a  definite  and  decisive 
style,  the  reader  being  told  just  what  to  do  in  every  emergency.  It  is  a  practical  book 
for  every  day  use,  and  the  large  number  of  excellent  illustrations  can  not  but  make  the 
treatment  to  be  pursued  in  any  case  clear  and  intelligible.  Physicians  and  nurses  will 
find  it  indispensible. 

Starr's  Diets  for  Infants  and  Children 

Diets  fcFOR  Infants  and  Children  in  Health  and  in  Disease. 
By  Louis  Starr,  M.  D.,  Consulting  Pediatrist  to  the  Maternity  Hospi- 
tal, Philadelphia.  230  blanks  (pocket-book  size).  Bound  in  flexible 
Morocco,  $1.25  net. 

Grafctrom's  Mechano-Therapy     JSSS^zSSL 

A  Text-book  of  Mechano-therapy  (Massage  and  Medical  Gym- 
nastics). By  Axel  V.  Grafstrom,  B.  Sc,  M.D.,  Attending  Physician 
to  the  Gustavus  Adolphus  Orphanage,  Jamestown,  New  York.  121110, 
200  pages,  illustrated.     Cloth,  $1.25  net. 

Sh^w  on  Nervous  Diseases  and  Insanity 

Recently  Issued — Fourth  Edition,  Revised 

Essentials  of  Nervous  Diseases  and  Insanity  :  their  Symptoms  and 
Treatment.  A  Manual  for  Students  and  Practitioners.  By  the  late  John 
C.  Shaw,  M.  D.,  Clinical  Professor  of  Diseases  of  the  Mind  and  Nervous 
System,  Long  Island  College  Hospital,  New  York.  121110  of  204  pages, 
illustrated.     Cloth,  $1.00  net.     In  Saunders"  Question- Compend  Series. 

Powell's  Diseases  of  Children       3d  Edition,  Revised 

Essentials  of  the  Diseases  of  Children.  By  William  M. 
Powell,  M.  D.  Revised  by  Alfred  Hand,  Jr.,  A.  B.,  M.  D.,  Dis- 
pensary Physician  and  Pathologist  to  the  Children's  Hospital,  Philadel- 
phia. l2mo  volume  of  259  pages.  Cloth,  #1.00  net.  ///  Saunders* 
Question -Com fend  Series. 


Saunders'  Compends 


SAUNDERS'  Question  Compends,  arranged  in  question- 
and-answer  form,  are  the  latest,  most  complete,  and  best 
illustrated  series  of  compends  ever  issued.  They  are  now 
recognized  as  the  standard  authorities  in  medical  literature 
with  students  and  practitioners  in  every  city  of  the  United 
States  and  Canada.  Since  the  first  appearance  of  these  in- 
valuable student-helps  there  have  been  sold  over  265,000 
copies.  The  entire  series  has  been  kept  thoroughly  revised 
and  enlarged  when  necessary,  many  of  them  being  in  their 
fifth  and  sixth  editions. 

A  COMPLETE  LIST  OF  VOLUMES 
Cloth,  $1.00  net  per  copy,  unless  otherwise  noted 

1.  ESSENTIALS  OF  PHYSIOLOGY.      2d  edition.       By  Sidney 

P.    BUDGETT,    M.D. 

2.  ESSENTIALS  OF   SURGERY.      7th  ed.     90  illustrations.    By 

Edward  Martin,  M.D. 

3.  ESSENTIALS  OF  ANATOMY.     7*  ed.      151   illustrations.     By 

C.  B.  Nancrede,  M.D. 

4.  ESSENTIALS  OF  MEDICAL  CHEMISTRY.   6th  ed.     By  Law- 

rence   Wolff,    M.D.     Revised   by  A.    Ferree  Witmer, 
Ph.D. 

5.  ESSENTIALS  OF  OBSTETRICS.    6th  ed.   75  illustrations.     By 

W.  Easterly  Ashton,  M.D. 

6.  ESSENTIALS  OF  PATHOLOGY  AND  MORBID  ANATOMY. 

By  H.  Harlow  Brooks,  M.D.     A  new  work.     Preparing. 

7.  ESSENTIALS  OF  MATERIA    MEDICA,     THERAPEUTICS, 

AND  PRESCRIPTION-WRITING.      7th  ed.     By   Henry 
Morris,  M.D.     Revised  by  W.  A.  Bastedo,  Ph.G.,  M.D. 

8,9.  ESSENTIALS  OF   PRACTICE  OF  MEDICINE.      By  W.    R. 
Williams,  M.D.     (Double  number,  $1.75  net.) 

(Continued  on  Opposite  Page) 


Saunders'  Compends 


10.  ESSENTIALS  OF  GYNECOLOGY.    6th  ed.    With  57  illustrations. 

By  Edwin  B.  Cragin,  M.D.     Revised  by  Frank  S.  Mathews, 
M.D. 

1 1.  ESSENTIALS  OF   DISEASES    OF   THE    SKIN.      6th  edition. 

61  illustrations.     By  H.  W.   Stelwagon,  M.D. 

12.  ESSENTIALS   OF    MINOR    SURGERY,  BANDAGING,  AND 

VENEREAL     DISEASES.       2d    ed.       78    illustrations.       By 
Edward  Martin,  M.D. 

13.  ESSENTIALS  OF  GENITO-URINARY  AND  VENEREAL  DIS- 

EASES.    By  S.  S.  Wilcox,  M.D.     A  new  work,  fully  illus- 
trated.   Just  Ready. 

14.  ESSENTIALS  OF  DISEASES  OF  THE  EYE. 

trated.     By  Edward  Jackson,  M.D. 

15.  ESSENTIALS   OF  DISEASES  OF  CHILDREN. 

Wm.  M.  Powell,  M.D. 

17.  ESSENTIALS  OF  DIAGNOSIS.      2d  ed. 

M.D.,  and  A.  A.   Eshner,  M.D. 

19.  ESSENTIALS  OF  NOSE  AND  TH 

By  E.   B.   Gleason,   M.D. 

20.  ESSENTIALS    OF  BACTERI 

and  6  plates.     By  M.  V 
Vogel,  M.D 

21.  ESSENTIALS    OF 

4th  ed.     53  ill 
by  Smith  F 

24.  ESSENTIA!  illus- 

trate 

25.  ESSr  Orations.     By 


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925  Walnut  St.,  Phila. 


